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1. EE 7 A E AE 7 ELADOM 12 Contacts G eneMachinoS ecsedi nrar serait rdias 7 LS The a de eo ae ou 8 2 ee ta 11 3 Points to consider before starting ce cceecccccsseeececeeeecceceeeeeeceeaeecessaeeeeeeeas 13 HiGro Installation aes nauscievsasauetedd seauautechestmaereannoueeaiay saw moawatanaestenceuiecedseanteseenstns 15 4 1 Preparing a space for the HIGTO cecccccccccccceeesseseeeeceeeeeseeessesseeeeeeeaaas 15 16 4 2 4 3 Unpacking the HOMO ne ss sedensse nee ee een eee sentence esse 16 4 4 Attaching the gas tank and tubing 17 AS POVNET COM COMMS C IONS en de D ed ee 19 4 6 Items to check prior to using the machine for the first 1816 19 Preparing SCO Wl DIS enie sesen NE NEE eee enter nn 21 21 0 a 22 2 ONG ONGOING 5 2 1 Cloning system and ROSES ne is aiaa rehita dan iieiea 22 MEd eere E EE EAEE EEEE 22 PSO VOS E A E 1 22 0 EE E s 24 2 o Makne Se ecra E E EER 24 24 0 0 UN RE 24 ed are 24 25 8 a a n 25 0 5 2 8 1 Dual temperature settings for a run ss 25 5 2 9 Humidity nee 25 De EP We MEI ee VOI ON Sn a O a 25 5 2 9 2 Dealing with excessive humidity 242 26 HiGro Shaker User Manual 0 Prepare the H ro or daly USS cn 27 6 1 Overview of tubing connections and gas F0W R dl
2. 27 6 3 Adjusting chamber temperature 29 6 4 Using the dual temperature feature 30 6 4 1 Overview of how the dual temperature feature 006178168 30 6 4 2 Setting the dual temperature feature for a run 30 6 4 2 1 Set the starting temperature 31 6 4 2 2 Set the idle final 10 sean rsenannattust taste anses i re iii 31 6 4 2 3 Setting the countdown time ss 32 6 4 2 4 Starting the dual temperature option for a run 32 6 4 2 5 Resetting the controller to default settings 32 DS LORS CAS 5 CU SS a da tan dde De 33 34 7 Starting and ending a run in the HAIG LO 8 35 e Hi Oee ie de tie di do 35 DZ NG D EE i E T P E E AE E E T 35 8 FAQS Frequently Asked Questions 37 a ae de ae 41 MATEO AU PODI a E 41 MeCh amMeAl Elect al PRO DIS an Ena 42 9 2 10 Quick guide to programming the Watlow temperature controllers 45 10 1 Reading or changing the temperature Setting 2 0 0 eceeeceeeecceeeesseeeeeeeeees 45 10 2 Making adjustments in the configuration menu cccceeeccecssseeeeeeeeeeees 45 LOS a de 46 10 4 Enabling the dual temperature feature 47 Work Less Do More About the HiGro 1 About the HiGro 1 1 ABOUT THIS MANUAL Please take the time to read this manual before operating the HiGro Though GeneMachines has included many safety features on the HiGro the inherent characteristics of the machine the fast rotational speeds and the gas fl
3. 1634 Problem Condensation is blocking the gas line and or flowmeter Suggestion s This can happen because of a couple of reasons a If the water bottle tips over water will enter the gas line and clog the flowmeter If this happens unscrew the tubing from the input fitting on the back of the HiGro and allow the tubing and or flowmeter to dry out b Sometimes condensation can build up in the exhaust lines leading from the chambers to the exhaust fitting on the back of the HiGro Generally this should not be enough to block gas flow However if gas is not flowing well it may be x GENEMACHINES Work Less Do More Troubleshooting necessary to unscrew the tubing fittings from the back of the chambers to let the tubing dry out for awhile If the con densation is building up too quickly for this solution try increasing the on time for the gas flow to help keep the air or oxygen moving through the tubing Problem The regulator valve opens gas flows into the water bottle and some water flows back up into the tubing towards the regulator Suggestion s This back flow occurs because there is a leak in the connections between the regulator and the water bottle If the back flow continues check the tubing fitting that attaches to the regulator If there is a leak it should be possible to feel some air coming out of the junction between the fitting and the tubing To fix a leak first try tightening the fitting If
4. GENEMACHINES Work Less Do More HiGro Shaker User Manual for all models HiGro Shaker Information Sheet Serial Number Date Installed GeneMachines Contacted Name HiGro Shaker User Manual 3 2000 2001 Genomic Instrumentation Services Inc All rights reserved No part of this publication may be reproduced transcribed transmitted or translated into any language in any form and by any means without the written permission of Genomic Instrumentation Services Inc Important notice GeneMachines believes that the information in this document is accurate The document has been carefully reviewed for technical accuracy In the event that technical or typographical errors exist GeneMachines reserves the right to make changes to subsequent editions of this document without prior notice to holders of the edition The reader should consult GeneMachines if errors are suspected INNO EVENT SHALL GENEMACHINES BE LIABLE FOR ANY DAMAGE ARISING OUT OF OR RELATED TO THIS DOCUMENT OR THE INFORMATION CONTAINED IN IT TRADEMARKS GeneMachines HiGro Shaker GeneMachines is a registered trademark of Genomic Instrumentation Services Inc in the U S and some countries HiGro Shaker is a trademark of Genomic Instrumentation Services Inc All rights reserved Watlow is a registered trademark of Watlow Electric Manufacturing Company Work Less Do More Table of Contents AIOT
5. C to 37 C within 55 minutes Uniformity Heating rate Timer function for dual temperature settings during a run Orbital 8mm for 96 well format models 4mm for 384 well format models Maximum 550 rpm or 650 rpm depending on model Resolution 1 rpm Air or Oxygen ready depending on model Flowrate control Range 0 30 SLPM or 0 6 SLPM depending on model Resolution 5 SLPM for large range flowmeter or 1 SLPM for small range flowmeter Controls for flowrate flow timing and delay to chambers Uncrated 26 L x 21 W x 22 H Supporting feet allow placement on a standard lab bench Crated 37 Lx 33 W x50 H Uncrated 1601b Crated 2801b 30 L x 22 W 40 H 10amp 120V 220V AC Samp 240 AC dedicated circuit with space for 2 plugs CE UL approved Motion control button for immediate shut off of rotation Balance indicator with automatic emergency shut off function to ensure proper operation Air or oxygen tank with safety restraints See Table 2 Air and oxygen tank specifications and recommendations on page 15 Work Less Do More specifications Components Sample Types Capacity Temperature Shaking Gas Flow Dimensions Weight Space reqs Power reqs Safety User supplies About the HiGro TABLE 1 Differences among the four HiGro models Specification HGA 02 96AL HGA 02 960L HGA 02 960H HGA 02 384A HGA 02 3840
6. 508 1634 Although the user may be able to help to diagnose the cause of this problem over the phone this problem is generally not user fixable Problem The HiGro has started making an unusual sound when shaking Suggestion s Please contact Customer Support at GeneMachines if this occurs Telephone 650 508 1634 There are two probable causes for an unusual sound Often the source of this is belt noise the belt has settled into a new position as an example and will not be a problem for using the machine However a problem with the drive train for instance a bearing failure can also cause changes in the sound that the HiGro makes This latter problem may require service by Genemachines Problem The HiGro will not shake properly or at all or has stopped shaking sometime during a run Suggestion s These problems usually require service by GeneMachines Unless there is an obvious reason for the slow shaking speed knob was turned down by accident or the stop the HiGro came unplugged or there was power outage we ask that users contact GeneMachines immediately Telephone 650 508 1634 HiGro Shaker User Manual 43 Work Less Do More Quick guide programming the Watlow temperature controllers 10 Quick guide to programming the Watlow temperature controllers These directions are taken from the Watlow Series 935A User s Manual that GeneMachines provides with the HiGro 10 1 READING OR CHA
7. The air regula 1 Attach the regulator set up to the gas 27 ar will look tank and use an adjustable wrench to D very similar tighten the fixture see Figure 4 1 Please p re note that the fixtures on air and oxygen reg ulators are NOT the same This is a safety measure to prevent attaching the incorrect ee The regulator regulator to the gas tank attach es tote FIGURE 4 1 Regulator set up gas tank here 2 Customers should be provided with two lengths of tubing one of which has a fer rule and hex fitting attached to both ends Tubing leading to the water This cord plugs into the back bottle attaches here panel of the HiGro a The tubing piece that has a fitting on BOTH ends will need to be cut to size using a sharp razor blade Measure out how much of this tubing piece is required to reach from the regulator set up which is now attached to the gas tank to the water bottle and then cut it to size with a sharp razor blade The water bottle will be secured onto the HiGro in a bottle holder see Figure 4 3 b If the fitting for the tubing on the regulator set up is covered remove FIGURE 4 2 Attaching the tubing the colored cap from the opening c Use the wrench to tighten the tubing end with the ferrule onto the reg Tubing end with ulator set up Figure 4 2 shows how the tubing and ferrule should look Oxygen ferrule attached 7 being inserted 3 The other end of this
8. between Chambers 2 and 3 This will help ensure that the HiGro stays balanced during the run Always fully secure chamber lids using all of the latches before running the HiGro Loose latches will damage adjacent chambers when operated Always leave the power cord connected to the HiGro and wall outlet for at least one minute after powering down In case of emergencies the mechanical drive system can be immediately shut off with either the motion button or power switch on the front panel The heating system can be immediately shut off with the power switch Always keep the area by the cooling fan clear of objects e While the HiGro is not being used do not leave the gas tank cylinder and main shut off valves open If they are open close the cylinder valve bleed off the excess gas by pressing the Test button on the pulse timer then close the main shut off valve e Never move carry the HiGro by pushing or pulling on any portion of the chambers If it is necessary to move the HiGro support the unit from the base and lift to move it Avoid slid ing the HiGro to move it A WARNING No smoking matches or open flames in the vicinity of an oxygen tank or the HiGro at any time WARNING When loading the HiGro the motion button AND power switch should be A off If the HiGro is accidentally turned on while loading the consequent shaking could cause serious injury to the operator and to the equipment HiGro Shaker User
9. is optimized in shallow well format The 4 0mm and 8 0mm shaking orbitals P described above will allow a vortex to form in each well during shaking This vortex is the key to creating vertical mixing in the well thus exposing more cells to the air above the well Furthermore the short well height means that the cells are mixed throughout the wells not just through a top layer This complete aeration prevents cell death and allows for optimal utilization of the available nutrients in the media Orbital For more information on this please refer to FAQS Frequently Asked Questions on page 37 HiGro Standard floor shaker Shallow well plates reduce production costs and increase throughput Standard shallow well plates flat bottom v bottom and u bottom are about a quarter of the cost of deep well plates the format traditionally used in cell suspension growth applications Furthermore in one growth run the HiGro can grow up to 4 608 samples in the 96 well shallow well format as compared with only 2 304 samples using deep well plates Shallow well plates are also widely compatible with various automation platforms to allow for easy downstream processing HiGro Shaker User Manual 21 5 2 GROWTH CONDITIONS We have compiled information from in house and customer testing to narrow in on the combination of parameters that achieves optimal growth in the HiGro Tables 4 and 5 show these protocol condit
10. the base of the chamber Make sure that the read out on the thermometer is facing upwards Set the chamber temperature to 37 C and attach the lid to the chamber Allow about 60 minutes for the chamber to heat up and equilibrate this time will vary depending on the starting Work Less Do More FAQS Frequently Asked Questions temperature Remove the chamber lid just enough to be able to shine a flashlight onto the thermometer and read the actual temperature To set the calibration offset Using the buttons on the temperature controllers enter the menu and scroll to the option If in the configuration menu the option is set to then the user will not have access to the menu To allow access change the option to be blank so that it is possible to enter the menu With the and E keys on the controllers choose the calibration off set in whole degrees Celsius Please refer to Quick guide to programming the Watlow temperature controllers on page 45 to learn how to navigate the menus HiGro Shaker User Manual 39 Work Less Do More 40 Troubleshooting 9 Troubleshooting 9 1 GROWTH PROBLEMS Problem Excessive evaporation of samples ie greater than 20 of the starting volume Suggestion s a Check to make sure that the tubing for the gas is routed through the water bottle see Figure 4 3 for a diagram of this set up and that the water level 18 enough 200ml to cove
11. the chamber lid b If all of the cassette shelves are being used try placing a couple of layers of paper towel on top of the cassette itself to absorb any drips Please keep in mind that if the paper towel is very thin or likely to get very wet it may sag and touch the top plate For these cases try wrapping and or taping the the towel around a piece of stiff cardboard and then plac ing it onto the cassette to catch the drips from the chamber lid Problem Cross contamination Suggestion s There are a few possible causes for well to well contamination a If the gas flow is too strong the liquid from one well may blow into adjoining wells Make sure that the regulator valve is not set above 10psi and that the flow rate level is not off of the flowmeter scale b Overfilling the wells or dribbling liquid on the tops of the wells can cause liquid to wick into adjacent wells Please refer to the first question answer under the FAQS section for suggestions on how to avoid these problems c For square well plates the shake speed may need to be reduced to avoid any splashing d Remember not to use hard plastic lids on the plates during growth runs e Exercise great care when handling full microwell plates Contact with the top surface of the cassette shelf can result in cross contamination f If cross contamination is persistent it may be necessary to use permeable tape over the plates Problem During a run the temperature goes abo
12. the cord leading from the sole 2 noid valve on the regulator setup into 4 He Outlet for the cord that connects the back panel of the HiGro see c to the valve on regulator set up Figures 4 1 and 4 4 3 Leave 4 to 6 inches of space between the back and left sides of the HiGro and any objects It is especially important not to block the fan Cooling fan In newer systems this is located on the back panel 4 6 ITEMS TO CHECK PRIOR TO USING THE MACHINE FOR THE FIRST TIME 1 Although unlikely it is possible that certain connectors on the HiGro will come loose during shipping Take a minute to look underneath each of the chambers if the lighting is poor a flashlight is helpful here Make sure that the HiGro is off and not plugged in while you do this Each chamber should have a white connector Push the connector ends together firmly Once the cords and tubing have been connected properly check to make sure that they will not rub against anything when the HiGro is running This is especially important for the power cord 2 Write the model and serial number for the HiGro unit on information chart provided at the beginning of this man ual The serial number is located on the back panel of the HiGro The number should resemble the following example HGA 02 D 062000 0075 If it is necessary to contact GeneMachines for service or support it is very helpful if the model and serial numbers are provided 3 If possible p
13. the leak persists it will be necessary to undo the fitting and check that the ferrule and washer on the tubing end are still there and intact Replacements of these should be in the extra items included with shipping of the HiGro If it is necessary to replace them be sure to cut the tubing just below the existing ferrule to create a new end See page 18 for directions on how to attach a new ferrule Problem The Balance Indicator light on the front panel turned on and the HiGro is has stopped Suggestion s Check to make sure that there 18 not an obvious reason that the Balance Indicator engaged These reasons can include a The HiGro was bumped or tilted for some reason during operation b When loading plates the chambers were not balanced properly or were not filled with a cassette See Load ing cassettes on page 33 for instructions on the correct way to do this Make sure that if there were any problems with the loading eg uneven distribution of plates into one chamber chambers without cassettes etc that these are corrected If the problem that triggered the indicator is not apparent try to reproduce the problem by turning the speed knob counter clockwise completely deactivating the motion button and turn off the power to the HiGro Then power on the machine activate the motion button and ramp up the speed If the Balance Indicator engages again please contact Customer Support at GeneMachines immediately Telephone 650
14. tubing will connect to the water bottle This attach into fitting ment will not require a ferrule however it is important to follow the directions below to get a proper seal on the tubing connection i Fitting with the a It will be necessary to slightly loosen the straight fitting on the bottle top ferrule still as opposed to the L shaped fitting attached b Insert the end of the tubing as far as possible into the straight fitting on the bottle top c Tighten the fitting with the wrench Tightening the fitting onto the tubing will squeeze the black sleeve in the fitting around the tubing and should form a tight seal d Test to make sure that the tubing attached properly by pulling gently on the tubing If the tubing end pulls out of fitting easily then the tubing end was not inserted far enough into the fitting It will be necessary to loosen the fitting again and try again 4 Use remaining portion of the cut tubing for connecting the water bottle to the input fitting on the back of the HiGro a In the same manner as described above attach the end of this tubing without the ferrule to the L shape fitting on the bottle b As with the fitting on the regulator set up it may be necessary to remove the colored cap from the IN fitting on the back panel of the HiGro HiGro Shaker User Manual 7 c Insert the other end of the tubing with the ferrule into the IN fitting and tighten with the wrenc
15. using oxygen with certain types of growth The delay timer will keep the pulse timer off until the set amount of time has elapsed During the delay time the green light on the face of the delay timer will blink When the delay timer is not in use the light will be on but should not be blinking a To set the delay turn the timer dial until the red marker points to the correct amount of time The units of mea sure for the time are indicated on the timer dial It is possible to change these using a small screwdriver to rotate the knob at the bottom left corner of the timer A Remember the delay time should be set at the start of the growth run unless the run is starting imme diately the delay should not be set at this time If the timer is set and then power to the HiGro is shut off it will be necessary to reset the delay timer To do this rotate it completely counter clockwise to 0 and then set the delay time again 11 Once the gas flow has been set the user has two options a If the HiGro is going to be used within a short time the pulse timer can be set for a very long off interval eg 45 minutes while the growth plates are being prepared b If the HiGro is not going to be used for a while then the user should shut the cylinder valve and bleed off the excess gas To do this close the cylinder valve completely and press the Test button until the hissing of the solenoid valve stops Then the main shut off valve should be clos
16. 0 40 and 45 minutes The pulse timer does not count gradations between these values 9 Adjustments to the gas flow rate can be made with the flowmeter located on FIGURE 6 5 Flowmeter diagram the left side of the HiGro a Press the Test button on the pulse timer shown in Figure 6 4 to see what Flowmeter reading level the flowmeter is set at currently is in SLPM b To reduce flow turn the knob clockwise to increase flow turn the knob Standard Liters counter clockwise see Figure 6 5 As with the output pressure changes in the Per Minute flowmeter levels will only be apparent when gas 1s flowing It may be necessary to make quite a few adjustments to the flowmeters to achieve the desired rates j Flowrate should See Table 4 for flow rate suggestions be measured by the center of the A PLEASE NOTE When setting the flow rate do not be concerned with flowmeter ball getting an exact level The flowmeter is only intended to regulate the flow Depending on the HiGro rate to an approximate level usually 1 SLPM Most growth applications Le 4 do not require an exact flowrate to work well shown here sa GENEMACHINES Work Less Do More Preparing the HiGro for daily use 10 When it is time to start a growth run the user will have the option to delay the start of gas flow using the delay timer on the front panel of the HiGro see Figure 6 4 A delay in gas flow is especially important when
17. CO2 build up 22 4 see also cell lysis condensation see chambers condensation on lids see flowmeter see plates condensation on covers see tubing cross contamination 22 37 41 cylinder valve see gas tank P pelleting see 6611 5 phage see cloning system plasmid see cloning system plate supports knob 33 34 rails 33 plates evaporation see humidity condensation on covers 22 37 covers 22 37 41 deep well plates 25 42 distributing among chambers 33 38 loading into cassettes 33 meniscus 23 37 round well vs square well 23 37 shallow well vs deep well 21 splashing 21 23 38 41 throughput 21 types 22 volume per well 22 37 power cord connection 19 rubbing 19 safety 13 19 power switch 13 35 protocols 96 well shallow well plates 23 24 PSI 28 pulse timer interval settings 23 24 28 42 Test button 13 28 35 R red knobs see plate supports regulator bleeding off gas see gas tank attaching to gas tank 17 fixture 17 main shut off valve 13 27 35 output pressure gauge 28 regulator knob 28 safety 13 17 RPM 35 run time overview 24 recommendations 23 24 5 sealing tape see air strips or plates covers shaking aeration see aeration balance indicator 34 43 noisy 43 orbital 21 25 recommended speeds 23 24 RPM 35 safety 13 speed 21 24 splashing 21 23 41 stopping properly 35 38 Work Less Do More lag phase 25 overgrowth 24 poor growth 42 protocols 96 well shall
18. Machines 935 Washington Street San Carlos CA 94070 USA hereby declares that under sole responsibility the products Hire high capacity shaking incubator for biological samples Model HGA 02 Are in conformity with the provisions of the following EC Directives including all amendments and national legislation implementing these directives Low Voliage Directive 724 REC EMC Directive 89 330 EEC And that the lollawing harmonized standards have been applied ENG1010 1 1993 A2 1995 ENG1326 1 1997 5501 1 1998 ENG O00 3 2 ENG 1000 53 34 CNG 1000 4 2 161 0000 4 3 ENGI O00 4 4 ENG 1000 41 35 ENG LOO0 d 6 ENGIO00 4 8 ENG 1O00 4 11 San Carlos CA 15 December 2000 Siunature Terry Allread Manager 0 deep well plates see growth or plates delay timer adjusting the range 29 recommendations 23 25 setting a delay 29 dimensions of the HiGro 8 E E coli see host electrical requirements 15 19 evaporation see humidity F fan cooling 13 19 feet Supports 15 16 ferrules see tubing fittings see chambers see tubing see water bottle flowmeter condensation in 42 flow path 27 flow rate settings 23 24 28 1 SLPM 28 testing flow rate 28 footprint of the HiGro 8 G gas flow bleeding off gas see gas tank chamber inlet valves see inlet valves delay see delay timer flowmeter see flowmeter manifold see manifold pulse timer see pulse timer settings see pulse timer or flowmeter tubing c
19. Manual 13 Work Less Do More 14 HiGro Installation 4 HiGro Installation 4 1 PREPARING A SPACE FOR THE HIGRO 1 The space requirements for the HiGro are 30 L x 22 W x 40 H Since the four feet that support the HiGro are inset from the sides of the machine it can be placed on a standard lab bench However it is important to keep two points in mind First the cassettes load into the HiGro chambers from the top If the HiGro is too high up it may be necessary to stand on a stool to properly load the cassettes Second for the sake of convenience we highly recommend that the HiGro be located next to preferably to the right of the gas tank that 18 supply ing it 2 To hook the HiGro up for gas flow the customer will need to provide the oxygen or air tank Please refer the the fol lowing table for information about what tank to purchase TABLE 2 Air and oxygen tank specifications and recommendations Specifications Air ready systems Oxygen ready systems Recommendations HGA 02 96AL amp HGA 02 384A HGA 02 96OL HGA 02 3840H Purity Zero Grade THC lt I ppm 99 6 minimum Recommended for regulator type dual stage also known as two stage dual stage also known as two stage Tank outlet connector CGA valve CGA 590 Female CGA 540 Male 250ft or 7m 250ft or 7m Safety Use safety restraints for attaching the tank to Use safety res
20. NGING THE TEMPERATURE SETTING Press and hold the TAN button for at least 5 seconds should appear in the read out Release EIP to return the read out to the current temperature reading gt If the read out displays ERA skip to Step 6 in this section to continue checking and setting the temperature e Ifthe read out DOES NOT display ie it continues to display a number proceed to the next step Press the up and down g buttons simultaneously for 3 seconds to enter the menu system Usually or will be the first displayed menu in the read out Press until FIN is displayed If WPTNA is not one of the available menus do not continue with this section The controller has not been set A up properly Refer to 10 4 Enabling the dual temperature feature page 47 for directions on how to do this Return to the beginning of this section once the controller has been set up Hold i and press until is displayed in the read out It will be necessary to ramp down through the temperatures past the minimum setting which is 200 C Once the read out displays AAA release GP Press and simultaneously for 3 seconds to get out of the menu system To check the set temperature press and hold 12h and for less than 3 seconds This will display the current set temperature in the read out Holding 5 for longer will display the read out To change the set temperature hold 22 and press or A to increase or decrease the temperature s
21. OR SET UP AND OR THE SWITCHING FROM OXYGEN TO AIR USE OR FROM AIR TO OXYGEN USE 3 Also required is a dedicated 10amp 120V AC 5amp 240V AC circuit with space for one plug As a safety precau tion plug the HiGro into a surge protected outlet strip before attaching to a wall outlet HiGro Shaker User Manual 15 4 2 ITEMS IN A STANDARD SHIPMENT 1 Wooden crate dimensions 37 L x 33 W x 50 H containing the HiGro itself and four plate cassettes stowed inside the HiGro chambers 2 Cardboard box dimenstions 15 L x 15 W x 21 H containing regulator set up water bottle power cords and customer care kit items which include tubing ferrules and this manual 4 3 UNPACKING THE HIGRO Do not throw away the wooden shipping crate or packing materials If for any reason it is necessary to send the HiGro A back to GeneMachines or any place the customer will need a crate for shipping In this case the customer is responsible for the cost of having a new crate built if necessary If possible it is best to store these items 1 To unpack the HiGro start by unlatching the fasteners at the bottom of each side of the crate 2 Once the latches are undone carefully lift the top portion of the crate straight up and off of the base Make sure that the crate top is complete clear of the HiGro before moving it aside This will require two or more people to do prop erly After the top has been remov
22. Plate type 96 well 96 well 96 well 384 well 384 well Shaking Speed Max 550 rpm 550 rpm 650 rpm 650 rpm 650 rpm Gas ready Air ready Oxygen ready Oxygen ready Air ready Oxygen ready HiGro Shaker User Manual 9 Work Less Do More 10 Labeled picture 2 Labeled picture FIGURE 2 1 HiGro picture Chambers 2 amp 3 off set Chambers 1 amp 4 off set and balance each other and balance each other Chamber 4 Chamber 3 Chamber lid f kal Chamber latch Chamber 1 Chamber 2 Water 20416 holder z Inlet valves C2 amp C4 not visible here m Flowmeter Inlet valves Plate support C1 amp C3 knobs Cooling fan Balance not visible indicator here Temperature controllers Gas flow delay timer Gas flow Speed knob pulse timer Power switch Speed read out in RPM Motion button revolutions per minute HiGro Shaker User Manual Ti Work Less Do More 12 Points to consider before starting 3 Points to consider before starting The HiGro is balanced in the following manner Chambers 1 and 4 off set and balance each other as do Chambers 2 and 3 Always run the HiGro with cassettes in each chamber to keep it balanced Try to divide the plates evenly between off setting chambers If for a growth run there are only enough plates to fill or almost fill one cassette divide the plates up between Chambers 1 and 4 or
23. again or if the HiGro does not start shaking please contact Customer Support at GeneMachines Tel 650 508 1634 u GENEMACHINES Work Less Do More 1 2 Starting and ending a run in the HiGro 7 Starting and ending a run in the HiGro 7 1 ACTIVATING THE HIGRO A It is assumed in this chapter that power to the HiGro is OFF and that gas flow rates have already been set when starting the following procedures After the cassettes have been loaded into the chambers open the gas inlet valves to each of the chambers that will be used if they are not already open See Figures 2 1 and 6 3 Depending on how the system has been left it may be necessary to open the tank and main shut off valves If these are already open and the gas inlet valves are open it may only be necessary to adjust the time intervals on the pulse timer Please refer to 6 2 Setting gas flow to chambers page 27 for complete instructions on how to do these steps If required set the delay timer knob for a delay in gas flow Usually this is only necessary for oxygenation with plasmid growth applications 4 Ensure that the speed adjustment knob is fully counter clockwise A 5 Ensure that the motion button is in the out stop position Turn on the power switch the temperature controllers should turn on and the speed read out should light up Depress the motion button such that the button lights up Upon activation a slow rotat
24. aker User Manual 25 2 If all of the cassette shelves are being used and excessive evaporation is still occuring place a flat piece of wetted paper towel in the lowest cassette shelf underneath the growth plate in this shelf Make sure that the paper towel in this case is very flat and that the plate 18 still level in the cassette 3 After each run remove the paper towel from the chamber cassette Otherwise after awhile an undesirable type of growth run may start to take over 5 2 9 2 Dealing with excessive humidity High humidity can cause water to condense on the underside of the chamber lid and then drip onto the cassette and top sample plate 1 If the cassettes are NOT filled to capacity with plates place an empty plate or plate lid in each of the top shelf posi tions to catch any drips from the chamber lid 2 If all of the cassette shelves are in use place a few layers of paper towel on top of the cassette itself to absorb any drips Make sure that the layered paper towel is thick enough to prevent it from sagging onto the top plate if it gets a little wet If the paper towels are likely to get quite wet try wrapping and or taping the towels around a piece of stiff cardboard before placing it on top of the cassette GENEMACHINES 26 Work Less Do More Preparing the HiGro for daily use 6 Preparing the HiGro for daily use 6 1 OVERVIEW OF TUBING CONNECTIONS AND GAS FLOW 1 The tubing for the gas sho
25. al They are off or closed when the knobs are vertical see Figure 6 3 Before a run make sure that the knobs of all the chambers that C1 Open are supposed to receive gas flow are in the open position 8 The pulse timer on the front panel of the HiGro controls the opening and shutting of the solenoid valve on the regulator see Figures 6 4 and 6 2 When the solenoid valve opens gas flows from the gas tank and regulator at the pressure shown in psi on to the output pressure gauge through the water bottle to the HiGro When the solenoid valve is open then a light turns on next to the on knob on the pulse timer The light next to the off knob will remain on as long as the valve is closed FIGURE 6 4 Delay and pulse timers on the front panel of the HiGro Test To set a delay in gas flow turn Valve cycle knob On time in seconds button the dial until the red marker points to the correct amount of delay time In this picture times are measured in hours To change the units of measure eg from hours to minutes use a screw driver to turn this knob in the appropriate direction Depending on the HiGro model the pulse timer may look different than the one pictured Valve cycle knob Off time in minutes a Adjust the gas flow cycle on the pulse timer by setting the cycle interval and duration The on times can be 0 5 2 4 6 8 and 10 seconds and allowable off times are 0 5 5 10 20 3
26. al temperature feature on the controllers vortex does not reach the base of the plate and com This is feature allows the user at the start of a run to program a plete mixing cannot be accomplished For this rea temperature change to occur at certain amount of time into the run as we coment ne D all possible However 11 deep well plates are abso lutely necessary then using a bead in each well may help achieve complete mixing 5 2 9 Humidity 5 2 9 1 Preventing evaporation It is important to maintain a high enough level of humidity in the chambers during a run to prevent excessive evaporation A certain amount of evaporation is to be expected TIP UNEVEN GROWTH up to 20 without adversely affecting growth If growth is especially uneven across a plate try the following techniques 1 When the chambers are mostly filled with growth plates the humidity should Use 8 less rich media or 8 mix of two be sufficient However if only a few plates are being grown wet a clean paper media e g 50 50 mix of LB and TB towel or small sponge with deionized H O and lay it flat in a cassette shelf that Reduce the shaking speed is below the lowest growth plate in the cassette It 15 best to place the paper towel or sponge inside of an uncovered one well plate e g Nunc OmniTray M as sec ondary containment and to prevent it from moving around during shaking Use a delay in gas flow 2 3 hours HiGro Sh
27. be transitioned to and optimized in a microwell format There are four HiGro models see Table 1 on page 9 for differences in the models All models have the following The HiGro unit has four growth chambers each holding one plate cassette The instrument comes with four cassettes customer chooses the types shallow or deep well Controls for chamber temperature shaking speed and gas flowrate and timing are located on the front panels of the instrument Also included are an air or oxygen regulator set up depend ing on model tubing and fittings a humidity bottle power cords User s manual which includes installation instruc tions See 4 HiGro Installation on page 15 and protocols See 5 Preparing growth plates on page 21 and a laminated Quick Start Guide Any sample type is acceptable within a 96 or 384 well microwell plate format depending on model HiGro is recom mended for growth applications but it is not limited to these types of samples Validated protocols are provided for stan dard E coli strain with plasmid and M13 cloning vectors See 5 Preparing growth plates on page 21 Limited to non corrosive and non volatile samples 48 shallow well plates or 24 deep well plates Independent temperature control for each chamber Regulated via four Watlow autotuning controllers Maximum 55 C Minimum Ambient 2 3 C Resolution 1 C Variability across plate lt 0 5 C Variability across chamber lt 1 3 C 22
28. bers and the user is growing cells with high copy plasmids for 16 hours the flowmeter should be set to 7 SLPM HiGro Shaker User Manual TABLE 5 Example protocol for growth in 384 wel Plasmid High copy eg pBluescript E coli robust strain eg DH5a DH10B LB Magnificent media 550 600 rpm 14 hours 4 chambers 4 7 3 chambers 3 6 2 chambers 2 5 1 chamber 1 3 0 5 seconds 0 5 minutes 37 C 5 2 4 Inoculum Variations in the level of inoculum will have a significant effect on the final growth yields shallow well plates 1 To achieve the best uniformity and repeatability inoculate with a measured dilution of a liquid cell culture Cloning System 2 For practical purposes inoculation using a robotic system will provide sufficient uniformity and is suitable to a high through Host put format Growth Media Shaking Speed 5 2 5 Shaking speed Total Run Time For Air use Flowmeter Level in SLPM Il 4 3 2 amp 1 cham is ber s in use 2 For unstable growth systems such as large insert cloning sys tems increasing the shaking speed may additionally stress the cells and cause poor yield or sample quality In these cases Gen Gas Off Time eMachines advises using lower speeds Temperature The shaking speed directly affects the rate of growth 1 In general the higher the shaking speed the faster the cultures Gas On Time The four flowmeter ranges sho
29. e ning If the chambers are rotating turn the speed knob fully counter clockwise press the motion button to deactivate and then turn off the power switch Cylinder Location of on off valve solenoid valve 3 Open the tank cylinder valve The tank pressure gauge should show an increase in pressure This gauge indi cates the pressure of the gas that remains in the cylinder When this gauge reaches zero the tank is empty Regulator a mN F d ar i ae m bent ee z knob Tubing leading to the water bottle attaches here T Cord plugs into the back Main shut panel of the HiGro off valve 4 Open the main shut off valve com pletely The output pressure should now increase HiGro Shaker User Manual 21 5 Adjust the output pressure with the regulator knob until it is 10 psi pounds per square inch It is only possible to see changes in output pressure when the gas is flowing 6 Press the Test button on the pulse timer to manually open the solenoid valve that FIGURE 6 3 Gas inlet valves control gas flow to the HiGro to check the output pressure see Figure 6 4 LA V A delay timer must be at the 0 zero position for the pulse timer to work Closed 7 Open the inlet valves to each of the chambers that will be used These on off valves that control the gas to each chamber are located either side of the HiGro The valves are on or open when the knobs are horizont
30. e remainder of the run 5 The user will need to reset the temperature controller back to the default standard settings after the run has com pleted 6 4 2 Setting the dual temperature feature for a run 1 Start by powering on the HiGro then follow the steps in the next five sections which correspond to the directions in Figure 6 7 The first three steps happen prior to the run The fourth step resets the controller to default settings after the completion of the run Resetting the controller to the default settings after a run See 6 4 2 5 Resetting the controller to default set tings on page 32 is very important in helping to prevent confusion for the next user Please do not skip this step If the 3 and my menus do not appear or if the timer does not behave as expected the dual tem perature feature has not been enabled properly Please refer to 10 4 Enabling the dual temperature feature page 47 for directions on configuring the controllers for the dual temperature feature Work Less Do More Preparing the HiGro for daily use FIGURE 6 7 Using the dual temperature feature The controller will display the current temperature of the chamber The red LED by 1 may be on This LED indicates that the chamber heat ers are on It comes on only if the chamber is below the set temperature Turn on power to the HiGro The red LED by 2 will be on This LED indicates that the contr
31. ecessary to clean the HiGro A If a spill occurs inside a chamber clean it up as best as possible with paper towels or dry cloths Then spray the inside of the chamber with 70 ethanol and wipe it with a clean paper towel or cloth If spills occur on the outside of the HiGro wipe them up as best as possible and then dampen a clean paper towel with ethanol and wipe the area where the spill occurred There are moisture sensitive parts inside the HiGro panels try to avoid letting drops fall into the machine 70 ethanol is also recommended for cleaning the cassettes The cassettes can also be autoclaved Cassettes should be laid on their sides in the autoclave Q I am concerned that the temperature read out on the temperature controller and the actual temperature in the chamber may differ if this is the case how do I correct for it A The best way to verify the actual temperatures in the chambers is to use a digital thermometer with an accuracy that is better than a half degree celsius If there is a discrepancy between the set and actual temperatures a calibration off set can be programmed to correct the difference To do the calibration offset it will be necessary to determine the required off set for the temperature controller Start by placing the digital thermometer inside an empty chamber Without allowing them to touch place the thermometer as closely as possible to the thermocouple wire which can be seen emerging up through
32. ed the HiGro will be sitting in a padded base It will require two or three people to lift and then move the HiGro out of the crate base To lift the HiGro it will be necessary to get your hands underneath the base of the HiGro Initially it may be difficult to get your hands between the foam padding and the HiGro base DO NOT resort to pulling on the HiGro chambers ONLY use the base of the HiGro for leverage A To prevent bodily injury or damage to the instrument never try to move the HiGro alone A Do not use any portion of the growth chambers for lifting or supporting the HiGro Always support the HiGro from the base of the instrument Avoid sliding the HiGro to move it 4 Once the HiGro is partially removed get a secure hold on the base of the machine and lift it to the location that you have prepared for it and set it down gently Again remember not to drag or push the HiGro along on the feet GENEMACHINES Work Less Do More 16 HiGro Installation 4 4 ATTACHING THE GAS TANK AND TUBING An adjustable wrench will be required for attaching the air or oxygen regulator to the gas tank and tubing Make sure that the gas tank has been secured into place prefera bly with safety restraints before attach s ing the regulator set up Never move the FR fg This picture gas tank with the regulator attached Location ofonoff ars iy 4 p pr 1 shows an oxygen solenoid valve RP UT regulator set up LE
33. ed as well The user should not have to make any adjustments to the gas flow when starting up HiGro the next time 6 3 ADJUSTING CHAMBER TEMPERATURE Once the HiGro is on the current temperature of each chamber should show on the corresponding temperature controller see Figure 6 6 FIGURE 6 6 Temperature controller 1 Press and hold the 3p button for at least 5 seconds 99 5 should appear in the read out Release 128 to return the read out to the current temperature reading a If 57 75 does appear continue with setting the temperature b A If DOES NOT appear in the read out ie the set temper ature continues to show it will be necessary to reset the controller to the default settings Please refer to 6 4 2 5 Resetting the controller to default settings page 32 for directions on how to this This is VERY IMPORTANT if the controller is not reset it will not be regulating the chamber to the expected temperature setting 2 To check the current setting press and hold for less than 3 seconds Longer than this time will cause MS to be displayed 3 To adjust the temperature setting press and hold 3 9 while pushing the up or down buttons to increase or decrease the temperature HiGro Shaker User Manual 29 6 4 USING THE DUAL TEMPERATURE FEATURE The Dual Temperature feature allows the user to set a temperature change to occur automatically after a certain amount of time This sectio
34. edures will only turn on the dual temperature feature for a temperature controller To use the dual temperature feature it will still be necessary to follow the steps in 0 4 Enabling the dual temperature feature page 47 Please note that the settings described for the configuration menu See 10 2 Making adjustments in the configuration menu on page 45 will also take care of enabling the dual temperature feature Press and simultaneously for 3 seconds to enter the menu system Press until is displayed Hold amp and press until B733 is displayed Release the 225 button Next press until is displayed Hold i and press until is displayed Release amp 15 P This enables the timer function and sets the units of measure to hours minutes Press until JA is displayed Hold and press until is displayed Release i Press until 200 is displayed Hold i and check that the setting is on Use or to change the setting if necessary Release 5 Press until is displayed Hold i and make sure that the display shows a 0 If it does not press or until 0 is displayed Release 5 Press and simultaneously for 3 seconds to get out of the menu system HiGro Shaker User Manual 4 1 2 3 10 11 12 Work Less Do More 48 Declaration of Conformity Genomic Instrumentation Services Inc dba Gene
35. em of poor aeration the cells are In general GeneMachines recommends growing too long and beginning to lyse The easiest solution to this is to stop the growth run sooner but for some labs where timing is essential reducing the shaking speed to slow the growth may be a more suitable option using air as opposed to oxygen for applica tions where gas waste build up over the growth wells is the main concern Further more if there is any uncertainty as to the effects of pure oxygen on the cells it 1s safest to use air GENEMACHINES Work Less Do More 24 Preparing growth plates 5 2 7 2 Oxygen 1 Oxygenation is very beneficial for M13 phage growth Growth comparisons show 8 3 to 5 fold increase in viable growth yield in the HiGro with oxygenation over growth in a standard floor shaker 2 For plasmid growth applications oxygenation is a double edged sword the right amount will improve viable yield too much will lead to no growth When cell concentration is low such as at the start of a growth run during the lag phase oxygen may inhibit cell growth To avoid this problem a delay 3 to 4 hours in the addition of oxygen during a run is highly recommended Since the main benefit of oxygen flow for plasmid growth is to prevent CO build up over the wells it is generally better to use too little oxygenation than too much 5 2 8 Temperature The most important thing to keep in mind for the temperature i
36. etting Once the temperature has been set release Sy 1 10 2 MAKING ADJUSTMENTS IN THE CONFIGURATION MENU Note This menu is pre set with the correct settings at GeneMachines The user should NOT need to readjust these settings Press and hold and simultaneously for 3 seconds to enter the menu system Press or until the display reads Press and hold 122 to show on the display read out and then use gt or until the display reads 141 then release EAN This will enter the configuration menu HiGro Shaker User Manual 45 1 2 3 4 Use the button to move through the configuration menu options At a menu option press to see the current setting To change the setting continue to hold and use the or Use the following set tings for the various menus Input type set to e Celsius Fahrenheit set to Input Range Low set to 5 degrees celsius Input Range High set to 55 degrees celsius Output 1 Function set to Output 2 Function set to Display Default set to Failure Mode set to e Timer Output Function LMI set to e Start Timer Function set to e Timer Ready Band set 0 Set Point Lock set to Lockout Tag set to blank 10 3 AUTO TUNING The auto tuning procedure enables the controller to self determine the optimal internal settings for the selected c
37. ex fitting and ferrule onto the tubing end see Figure 4 2 The fitting and ferrule may fit somewhat tightly onto the tubing so it may require quite a bit of pressure to slide them in place c After the hex fitting and ferrule are on the tubing screw the fitting into the OUT opening by hand d Once the fitting is hand tight use a wrench to tighten the fitting an additional 1 and 1 4 turns To help deter mine how far this is mark one of the sides of the fitting before starting the rotations e Unscrew the fitting completely The ferrule should now be squeezed around the tubing f Now the fitting can be tightened onto the back of the HiGro with a wrench as described for the other attach ments PLEASE NOTE Any time it is necessary to use a new ferrule it will be necessary to follow the directions given A above to secure the ferrule onto the tubing GENEMACHINES Work Less Do More HiGro Installation 4 5 POWER CORD CONNECTIONS There are two outlets on the back panel of the HiGro see Figure 4 4 One outlet is for the HiGro power cord the other y Housing Outlet for main connects to the solenoid valve on the LOr RISE HiGro power cord regulator set up FIGURE 4 4 Back panel of the HiGro 1 Attach the power cord provided with the instrument from the HiGro to a surge protector outlet strip and attach this to the wall outlet a dedi cated 10amp 120V AC 5amp 240V AC circuit 2 Plug
38. h Make sure that the tubing has at least one loop in it between the bottle and the input fitting The loop will help prevent water from entering the HiGro Check that none of the tubing is rubbing against any moving parts on the HiGro FIGURE 4 3 Tubing attachments Looped tubing helps prevent any water droplets from getting into valves on HiGro Tubing between HiGro and botte should enter bottle INPUT OUTPUT through L shaped fitting Tubing inserted a Route to 5 into underside mU LT ventilated areg 0 0 t should These fittings are on the back extend into panel of the HiGro The oxygen e water eat peed E mi tubing enters and exits the HiGro a H 0 through these fittings tt 5 The other portion of tubing provided with the HiGro is for the exhaust from the machine If the HiGro will be used for M13 phage growth or any application involving large amounts of oxygen GeneMachines recommends attaching the exhaust line and routing it to a ventilated fume hood Generally an exhaust line is not necessary for plasmid growth appli cations Whether or not the exhaust tubing will be attached remove the plastic cap if present from the OUT fix A ture on the HiGro If this cap is left on it can cause build up of CO in the growth chambers a To attach an exhaust line start by removing the ferrule and fitting if present from the OUT fixture on the rear panel of the HiGro b Slide a h
39. hamber avoiding forward or backward motion see Figure 6 8 Do not allow the plates to catch against the plate supports this will cause samples to splash Please note that the HiGro must be balanced to operate properly Chambers 1 and 4 off set and balance A each other as do Chambers 2 and 3 To keep the machine balanced during operation each of the chambers should be loaded with a cassette whether or not the chamber is going to be used for cell growth It is also important to try to load plates evenly between off setting chambers Remember that if there are only enough plates to fill or almost fill one cassette for a growth run divide the plates up between Chambers 1 and 4 or between Chambers 2 and 3 The same principle is true if there are only enough plates to fill about three cassettes HiGro Shaker User Manual 30 FIGURE 6 8 Inside view of a chamber and loading a cassette into a chamber Gas manifold lt Chamber lid latch Plate support rails While loading the cassette do not allow the plates to catch against the plate supports This will cause samples to splash Plate support knob 6 Once the cassette is resting in the chamber flip the plate support knob down This will secure the cassette and plates in the chamber It is very important to remember to do this because improper securing of the sample plates will lead to sample splashing during a growth run Furthermore if the cassettes hit against
40. he countdown timer the dual temperature option is ready to be used 1 In the temperature read out screen ie not in the menu system press the button ONCE to start the count down timer 2 To stop the dual temperature feature press the button a second time At this point the controller will no longer be timing and the temperature will be regulated to the idle final temperature setting Please note Anytime that the controller is set to an idle final temperature as opposed to 1 13 and the countdown timer is not running the controller will regulate to the idle final This idle final temperature is NOT the same as the start temperature unless they have been set to the same value Remember that pressing 59 from the temperature read out screen ie not in the menu system will display the start temperature setting not the idle final temperature setting 3 To start the dual temperature feature again press the button again The controller will start counting down the full time It will not start where it left off last 6 4 2 5 Resetting the controller to default settings 1 Press and simultaneously for 3 seconds to enter the menu system 2 Press until 7 is displayed 3 Hold amp 3 and press until is displayed in the read out It will be necessary to ramp down through the temperatures past the minimum setting which is 200 C Once the read out displays FA release Gap 4 Press and simultaneously for 3
41. ibed in 6 3 Adjusting chamber tem perature page 29 2 To check the starting temperature press the amp 3p button 6 4 2 2 Set the idle final temperature The idle final temperature is the setting that the controller will switch to after the countdown time has finished 1 Press and simultaneously for 3 seconds to enter the menu system 2 Press until 7 is displayed HiGro Shaker User Manual oi 3 Set the final temperature also refered to as the idle temperature by holding the 59 button and pressing either E or A The temperature setting is in degrees Celsius Release the 38 button when finished 4 Press and simultaneously for 3 seconds to get out of the menu system 6 4 2 3 Setting the countdown time 1 Press and simultaneously for 3 seconds to enter the menu system 2 Press 53 until par is displayed 3 Hold 39 and press or until the desired countdown time is displayed and then release the 39 but ton The time will be in hours minutes As two examples a 5 minute timing would appear as 00 05 and a 5 hour timing as 05 00 This countdown time determines how long the chamber will remain at the starting temperature before changing to the idle final temperature 4 Press and simultaneously for 3 seconds to get out of the menu system 6 4 2 4 Starting the dual temperature option for a run After setting the start and idle final temperatures and t
42. ion of the chambers is normal Slowly turn speed Knob to desired setting The indicated value on the read out is in RPM revolutions per minute Check the time intervals on the pulse timer and the level on the flowmeter to ensure that these are set correctly 5 N 7 2 ENDING A RUN Once the run is finished close the cylinder valve Bleed the remaining gas in the system by pressing and holding the Test button until the hissing finishes Close the main shut off valve Turn down the speed knob to full counter clockwise position When the speed panel meter reads nearly zero and motion has nearly stopped push the motion button to stop the rotation Power down the HiGro prior to unloading HiGro Shaker User Manual 35 Work Less Do More FAQS Frequently Asked Questions 8 FAQS Frequently Asked Questions Q How full should I be able to fill the plate wells A The high surface tension of the samples in each well and the small shaking orbital should allow the user to fill the wells almost to the top without seeing any spill over from adjacent wells For a list of maximum volumes in various plate types please see Table 3 It is important to keep in mind a few things a The meniscus of the liquid in the well should form a U below the top rim of the well as opposed to bulging up out of the well see Figure 5 2 b It is important not to dribble liquid up over the edges
43. ions for some of the most commonly used cloning systems when grown in a robust E coli host strain Unfortunately cell growth is not a cookie cutter process fully optimizing growth may involve adjustments to these protocols GeneMachines recommends trying these protocols first and then making changes if necessary 5 2 1 Cloning system and host With the multitude of cloning systems and host strains it is not feasible to provide protocols for all of the possible combinations Since the majority of high throughput applications have been adapted to work in a few robust strains of E coli GeneMachines has focused on developing protocols for this host system Customers do sometimes report to GeneMachines on their successes failures at growing other types of strains and vectors in the HiGro If questions about other applications arise please contact GeneMachines to find out about any suggested growth conditions in these cases TIP DON T COVER PLATES 5 2 2 Growth media One of the biggest advantages of the HiGro is the capability for The unique features of the HiGro definitely help improve oxygenation during growth This helps prevent CO build up results but the key variable for yield is the growth media over the wells Covering the plates will counteract the benefits of using air or oxygen GeneMachines strongly recommends 1 Super rich media such as Terrific Broth TB and YT can potentially support a very high growth rate and cell yie
44. l 96 u bottom circular well 384 flat bottom rounded square well 384 flat bottom circular well 384 v bottom circular well Plasmid Low copy E coli robust strain 50 50 LB TB mix 480 500 rom 18 hours All plates are shallow well types M13 Phage E coli robust strain such as DH12S 2xTB 600 rom 18 hours Preparing growth plates No matter what type of plate is used customers should use the fol lowing guidelines in determining a good sample well volume The meniscus of the liquid should form a U just below the top rim of the well as opposed to bulging up over the rim see Figure 5 2 Some deep well plates have square wells To avoid risk of splashing in plates with square wells we would advise not filling the wells as close to the top of the plate and reducing the shaking speed If the growth media contains any detergents that significantly reduce the surface tension of the liquid it may be necessary to reduce the growth volume and to reduce the shaking speed TABLE 4 Example protocols for growth in 96 well shallow well plates BACs E coli robust strain such as DH10B LB 420 rom 18 hours Plasmid High copy E coli robust strain eg DH5a DH10B 1xTB YT 500 550 rpm 16 hours Cloning System Host Growth Media Shaking Speed Total Run Time The recommended flowrates are given as ranges because it is not crucial to use exact flowmeter leve
45. ld These are good choices for stable cloning systems with small sized inserts lt 10kb against using lids or tape to cover the plates If covering the plates is a necessity try using a permeable sealing tape eg air strips or normal sealing tape with puncture holes for each well Since condensation may build up on the tape dur 2 For cloning systems with large inserts the rapid growth ing the run make sure that each well is completely sealed off rate may cause problems e g kicking out the plasmid from adjoining ones to prevent wicking and cross contamination These systems are better suited to growth in a less rich media such as Luria Bertani LB 5 2 3 Growth volumes As shown in Figure 5 1 the HiGro uses a small shaking orbital This combined with the high surface tension of the samples in each well allows the user to fill the wells almost to the top without lt seeing any spilling from adjacent wells ONE j Rim of the well 1 Appropriate maximum volumes for different plate types are given in Table 3 Please keep in Empty well mind that these amounts are generalized for over all plate designs variations between different brands may affect the maximum usable volume FIGURE 5 2 Filling sample wells 7 GENEMACHINES Work Less Do More TABLE 3 Growth volumes Volume ul Plate type 96 Flat bottom circular well 300 280 250 96 v bottom circular wel
46. ls 4 chambers 8 9 3 chambers 6 7 2 chambers 4 5 1 chamber 1 3 4 chambers 4 5 3 chambers 3 4 2 chambers 2 3 1 chamber 1 2 0 5 seconds 0 5 minutes 3 hrs for Os 37 C Chart last updated 04 25 2001 23 NA 4 chambers 15 17 3 chambers 11 13 2 chambers 8 10 1 chamber 4 6 4 seconds 0 5 minutes none 37 CT 4 chambers 8 9 3 chambers 6 7 2 chambers 4 5 1 chamber 1 3 4 chambers 4 5 3 chambers 3 4 2 chambers 2 3 1 chamber 1 2 0 5 seconds 0 5 minutes 3 hrs for Os 37 C 4 chambers 12 14 3 chambers 9 11 2 chambers 6 8 1 chamber 3 5 4 chambers 4 6 3 chambers 3 5 2 chambers 2 4 1 chamber 1 3 0 5 seconds 0 5 minutes 3 hrs for Os only 37 C For Air use Flowmeter Level in SLPM 4 3 2 amp 1 cham ber s in use For Oxygen use Flowmeter Level in SLPM 4 3 2 amp 1 cham ber s in use For both Gas On Time For both Gas Off Time For Oxygen use Gas Delay Temperature The maximum shaking speed is 550rpm or 650rpm depending on model Air flow settings do NOT apply to M13 phage growth For this type of growth GeneMachines recommends using oxygen to achieve best results Please note that there are four different flowmeter ranges shown for each cloning system These represent the different flow rates when using four three two or one chambers for growth For example if gas is shut off to two out of the four cham
47. mperature controllers adjusting configuration settings 45 auto tuning 46 error messages 42 fluctuations in temperature 25 pre heating chambers 33 programming quick guide 45 setting a callibration off set 38 setting chamber temperature 29 45 tuning parameters 41 46 throughput 21 tubing IN OUT fittings 18 27 condensation 18 42 connection to regulator 17 connection to the HiGro 17 connection to water bottle 17 exhaust from HiGro 18 ferrules 17 fittings 17 leaks 43 new ferrule fitting attachment 18 overview of connections 27 water back flow 43 U uneven growth 25 unpacking the HiGro 16 V valve timer see pulse timer valve see solenoid valve vortex see aeration W water bottle see also tubing back flow from 43 connection to regulator 17 connection to the HiGro 17 fill volume 18 27 41 fittings 17 Overview of connections 27 Work Less Do More Work Less Do More J4 Work Less Do More Work Less Do More
48. n describes how to set the temperature change and timing PLEASE NOTE When using the dual temperature feature for the first time it is very important to check that A the configuration settings are correct Please refer to 10 4 Enabling the dual temperature feature page 47 6 4 1 Overview of how the dual temperature feature operates This feature is set up such that once it has been enabled and set properly for a temperature controller the following steps should occur 1 Once the user starts the countdown timer for the dual temperature function the HiGro chamber will be regulated to a start temperature e This start temperature can be checked by pressing 3 2 The chamber will remain at the start temperature for the duration of the countdown time which has been set by the user e This countdown time can be checked by pressing and simultaneously for 3 seconds to enter the menu system Usually or will be the first displayed menu in the read out Use or to reach the Fans menu Hold 8 to display the countdown time in hours minutes 3 Once the countdown time elapses the chamber will be regulated to the idle or final temperature e This idle final temperature can be checked by pressing and simultaneously for 3 seconds to enter the menu system Use or g to reach the FEJN menu Hold 3 to display the idle final temperature 4 The chamber will remain at the idle final temperature for th
49. of the wells Dribbles will allow the liquid to wick from one well into the next This sometimes becomes a problem if the media is dispensed into the wells using an automate plate filler On occasion these instruments will leave drops on the tops of the well walls It may be necessary to blot the tops of the plates with a sterile absorbent towel if dribbles occur c Another point to consider is the shape of the wells in the plates that are being used 96 well shallow well plates tend only to be round whereas some 96 deep well plates have square wells Although it is not certain that splashing occurs in square well plates it seems probable that it could happen for 96 well plates It is not advisable to fill square wells as close to the top of the plate Reducing the shaking speed will also help prevent splashing For the 384 well square well plates the bigger problem than splashing may be that no mixing occurs In square wells the surface tension of the sample may be too high to allow the liquid to move d If the growth media contains any detergents that significantly reduce the surface tension of the liquid it may be neces sary to reduce the growth volume and to reduce the shaking speed 6 Should I use lids on the microwell plates when I use the HiGro A No Since the chambers are heated condensation will build up on the plate lids The shaking will cause this liquid to wick over and into adjoining wells and thus lead to cross con
50. oller is currently regulating the temperature to the idle final temperature 2 1 Set the idle final ee and countdown timer 8 Set the start Start the count temperature p 5 D 3 3 down timer ind Press gt and Co press or 6 8 and hold for Press ED or E Hold 3 and press gt to set the 3 seconds to enter until the 783 menu or to set the menu system shows the idle temperature In the temperature read out screen ie not in the menus press ature _ down arrow CD 03 30 c tran button ONCE to 30 2 Press and start the timer SET gt and hold for Hold 355 and then Release and then ag 5 3 seconds to exit press 3 OT 9 press 3 or 9 5 Onn the menu system to set the countdown until the pay menu 3 time hours minutes shows Start temper 4 After the run Return the controller to the default set up for other users Ch ILE j JD C C Press and Press amp and gt and hold for Piss lt gt or Hold 7 4 press 5 down arrow gt and hold for 3 seconds to enter until the until the read out says pF Eric 3 seconds to exit the menu system shows will be necessary to go past 200 C the menu system 6 4 2 1 Set the starting temperature The starting temperature is the temperature setting that the controller will be at during the countdown period 1 The procedure for setting this starting temperature is exactly the same as descr
51. onfiguration options It is not necessary to re tune the controllers on a regular basis However if the controller or heating system for a chamber has had any problems that required service GeneMachines recommends auto tuning after the repair Furthermore if users notice any unusual inaccuracies in a chamber temperature controller eg fluctuation shooting past a set point etc GeneMachines recommends auto tuning as a first step in correcting the problem Please note that the auto tuning process will over ride any calibration off sets that may have been set up during testing at GeneMachines or by other users If a calibration off set is required for a chamber please refer to Chapter 8 page 37 for directions on setting it 1 Press 6 and simultaneously and hold for 3 seconds to enter the menu system 2 The display should now read If it does not scroll to this menu using the button 3 Press and hold i 3 and select 1 35 on the display using the button will flash in the display read out When auto tuning is complete the display will return to normal 4 To stop auto tuning press and hold and select on the display using the down button Note Auto tuning can take up to 85 minutes but usually takes less than 45 minutes g GENEMACHINES Work Less Do More Quick guide programming the Watlow temperature controllers 10 4 ENABLING THE DUAL TEMPERATURE FEATURE These proc
52. onnections see tubing flow path 27 overview 24 oxygen vs air 24 setting up 27 37 gas tank attaching regulator 17 bleeding off excess gas 13 29 bleeding off excess oxygen 35 cylinder valve 13 27 35 pressure gauge 27 safety 13 17 glass beads see beads green knobs see inlet valves growth aeration see aeration media see media run time see run time volume see plates volume per well cell lysis see cell lysis deep well plates 25 42 HiGro Shaker User Manual Di Numerics 384 well plates see plates 96 well plates see plates A aeration 21 24 25 42 air regulator see regulator safety 15 tank specifications recommendations 15 air strips 22 37 41 see also plates covers B balance indicator 34 43 balancing chambers see chambers beads 25 42 see also growth deep well plates C cassettes balancing 13 33 38 43 cleaning 38 loading into chambers 33 loading plates 33 shelf 33 cell death see cell lysis cell lysis 21 24 42 chambers cleaning see cleaning the HiGro inlet valves see inlet valves manifold see manifold temperature see temperature controllers balancing 13 33 38 43 condensation on lids 41 gas fittings 27 latches 13 42 lids 13 34 42 loading cassettes 13 33 power connectors 19 pre heating 33 preventing damage to 13 16 safety 13 setting temperature 29 cleaning the HiGro 26 38 cloning system insert size 22 24 overview 22 oxygen vs air 24 types 23 24
53. oss in chamber temperatures Lift the cassette holders as steadily as possible avoiding forward or backward tip ping of the cassette The chambers should be set at the 4 Load the plates into the cassettes Push each plate to the back of the cassette behind the front lip of the shelf The back of the plate should slide over the back lip of the shelf If the cassette is not going to be loaded to capacity distribute the plates If only a few plates are being grown evenly between the top and bottom of the cassette Distributing the plates will pro in each of the chambers place a one vide a more secure and stable anchoring of the entire cassette within the chamber well plate with a dampened paper towel or sponge wet with dH O in 5 Reload the cassette into the chambers As mentioned above it is best to be posi tioned above the chambers for this procedure Slowly guide the loaded cassette into the growth chamber reversing the removal procedure Maintain an even pressure against the back edge of the chamber the lowest shelf of the cassette For more information on preventing evap oration see Humidity on page 25 Make sure that the fronts of the cassettes are facing the plate supports when loaded into the chambers This is the only correct way that the cassettes fit into the HiGro chambers Try to keep the cassette shelf tab roughly centered between the plate support rails Insert the cassette straight into the c
54. ost the HiGro Quick Start Guide and manual near the unit HiGro Shaker User Manual 19 Work Less Do More 20 Preparing growth plates 5 Preparing growth plates 5 1 BACKGROUND The HiGro combines a unique set of capabilities which facilitate improved growth yield for high throughput recombinant DNA applications To take advantage of the benefits that the HiGro can provide GeneMachines has compiled some example protocols in this section This is not to say that customers should be limited to these parameters the HiGro has potential for success with a wide variety of growth applications These other applications may require very different parameters for optimization One very important factor to the success of growth in the HiGro is the plate format that is used Although the HiGro will hold any plate with a standard microwell footprint GeneMachines recommends using 96 or 384 well shallow well plates depending on model The reasons for this are three fold The shaking orbital matches the well diameter of a 96 or 384 well plate Unlike standard floor shakers which rotate on an orbital of 25mm the HiGro rotates on one of two smaller orbitals depending on model 8mm or 4mm These orbitals are just slightly smaller than the standard well diameters in 96 and 384 well plates respectively thus enabling the use of a very high shaking speed without causing splashing FIGURE 5 1 Mixing in the wells Aeration
55. otion button to stop the shaking stresses the drive motor and will reduce the life of the motor Furthermore if there are samples in the HiGro this abrupt motion may cause them to spill The motion button is a safety device If there is an emergency that requires that the HiGro be stopped immediately definitely use the motion button to stop rotation Otherwise to stop the HiGro turn the speed knob fully counter clockwise wait until the HiGro has stopped moving or 1s just barely rotating and then press the motion button Q If I am only runing a few plates in the HiGro do I need to distribute them evenly amongst all of the chambers A Yes and no If there are enough plates to fill or almost fill a cassette GeneMachines STRONGLY RECOMMENDS dividing the plates between Chambers land 4 or between Chambers 2 and 3 The same is true if there are almost enough plates for three cassettes in this case divide the plates evenly among all four chambers Dividing up the plates will help reduce wear on the drivetrain NO MATTER WHAT it is crucial to run the HiGro with a plate cassette in each chamber at ALL times even if there are no plates in some of the cassettes This is essential for keeping the HiGro balanced and stable REMEMBER to secure all of the cassettes with the plate supports before running the HiGro If the cassettes are loose during a run this could also cause the HiGro to get out of balance QO What cleaning agents should I use if n
56. ow make it impossible to completely safe guard the user or the HiGro itself from harm For this reason it 18 especially important for anybody using the HiGro to be familiar with the information in this manual Throughout this manual especially important information will be marked by a A To avoid causing any harm to yourself others or the HiGro please take note of these sections Manual Version HGA 02E 012002 1 2 CONTACTING GENEMACHINES Please contact GeneMachines immediately if there are questions about operating and maintaining the HiGro or if service 1s required To help us assist you please include or be prepared to provide the following information when contacting GeneMachines e The unit serial number located on the back panel of the HiGro The name of the organization or institution that you are from e Your contact information A description of the problem GeneMachines contact information Phone 1 877 855 GENE 4363 Fax 1 650 508 1644 Email support genemachines com Address HiGro Customer Support 935 Washington Street San Carlos CA 94070 HiGro Shaker User Manual 1 1 3 THE HIGRO SYSTEM The HiGro is a high capacity incubating shaker for microplates 96 or 384 well depending on model that combines a small shaking orbital gas flow and temperature controls The HiGro is an ideal solution for laboratory facilities with high throughput growth needs and or applications that need to
57. ow well plates 23 24 uneven growth 25 42 yield 21 25 H host E coli growth temperature 23 24 25 growth temperatures 25 overview 22 types 23 24 hotels see cassettes humidity Overview 25 preventing evaporation 25 33 41 inlet valves flow path 27 opening and closing 28 35 inoculum 24 installing the HiGro 15 inter val timer see pulse timer L latches see chambers lids see chambers 13 phage see cloning system M main shut off valve see regulator manifold 27 34 media detergents 23 37 recommendations 23 24 42 types 22 23 24 microtiter plates see plates microwell plates see plates mixing see aeration motion button 13 35 38 moving the HiGro 13 16 N noises while in operation 43 O orbital see shaking ergrowth 24 see also cell lysis ergrowth see growth cell lysis ygen delay see also delay timer regulator see regulator delay 23 25 29 42 safety 13 15 tank specifications recommendations 15 OV OV Ox 12 SLPM 28 see also tubing solenoid valve Watlow see temperature controllers connection to the HiGro 17 19 wicking see cross contamination overview of function 28 wooden crate space requirements 15 19 dimensions 16 speed knob 27 34 35 38 unpacking 16 speed read out 35 spills 38 splashing see plates or shaking strain see host tank see gas tank temperature E coli growth temperature 23 24 25 overview 25 te
58. r the end of the tubing that hangs down into the bottle b If the gas flow rate is set very high or the on time for the gas flow is very long then the gas flow could be causing added evaporation Lowering shortening these parameters may be necessary c Another thing to consider is the number of plates being grown in a chamber If only a few plates are being grown wet a clean paper towel or small sponge with deionized H O and lay it flat in a cassette shelf that is below the lowest growth plate in the cassette It is best to place the paper towel or sponge inside of an uncovered one well plate e g Nunc OmniTray as secondary containment and to prevent it from moving around during a run d If all of the cassette shelves are being used and excessive evaporation is still occuring place a flat piece of wetted paper towel in the lowest cassette shelf underneath the growth plate in this shelf Make sure that the paper towel in this case is very flat and that the plate is still level in the cassette Problem Excessive condensation forms on the insides of the chamber lids during runs and drips onto the plates Suggestion s Unfortunately there is no easy and or neat way to reduce the humidity in the chambers on a regular basis To solve this problem it is best to fix the symptoms a If the cassettes are not filled to capacity with plates GeneMachines recommends placing an empty plate in each of the top shelf positions to catch any drips from
59. s that the HiGro can only heat the chambers there are no cooling mechanism TIP THE TRUTH ABOUT BEADS Once a chamber reaches the programmed temperature the heaters willturn Many HiGro customers ask about the use of beads off until the temperature drops just below this set level soda lime glass beads in the wells to enhance growth While beads do improve mixing when 1 If the chambers are completely filled with growth plates during a growing plates in a standard floor shaker they are run the temperature in the chamber may climb one to two degrees usually unnecessary when using the HiGro above the set temperature Most E coli strains grow at 37 C but are The HiGro has a shaking orbital that matches the not too sensitive to minimal temperature changes However some diameter of a well in a 96 or 384 well plate altered strains may suffer heat stress at 38 C For these strains set the depending on model This means that at a high temperature one degree below the standard temperature to prevent this shaking speed a vortex can form in the sample well from occuring without causing spilling The vortex will achieve the vertical mixing the key to good aeration and thus enhance growth 5 2 8 1 Dual temperature settings for a run The exception to this occurs when using deep well Customers that have growth applications that require two temperatures plates in the HiGro if the wells are filled up the during a growth run can use the du
60. seconds to get out of the menu system GENEMACHINES Work Less Do More Preparing the HiGro for daily use 6 5 LOADING CASSETTES 1 1 Turn off the power to the HiGro before loading cassettes TIP BEFORE STARTING 2 Open the incubation chamber by loosening the three latches that secure the 1 If the gas flow settings have not been chamber lid Remove the chamber lid made prior to this point please read Setting gas flow to chambers on page 27 Always set the gas flow prior to loading cell samples 3 Remove the cassettes from the incubation chambers by flipping up the red knob on the front of the chamber This will move the cassette support inside the chamber such that it is no longer pressing against the plate cassette see Figure 6 8 for pictures of these items For loading and unloading the cassettes it Ai igo required temperature and preheated is best to be positioned above the chamber tops so that it 1s possible to see the cas before loading the cassettes Preheat sette top in the chamber this might require a stepping stool Take hold of the ing the chambers can take up to 60 cassette top or handle While applying an even and continuous pressure minutes Since power is cut off while against the back of the chamber slowly lift the cassette out of the chamber loading the chambers there might be a Try to keep the cassette shelf tabs roughly centered between the plate holder rails slight l
61. tamination Furthermore the lids will prevent oxygen from reaching the cells Q Should I use tape over the plates this tape is sometimes called air strips or sealing tape A GeneMachines strongly recommends against using tape to cover the plates If it is necessary to cover them GeneMachines suggests using permeable tape or tape in which holes have been punctured for each well Keep in mind however that the tape will prevent as much air or oxygen from getting into the wells as does when the plates are uncovered This in turn will allow carbon dioxide to build up over the wells and thus slow or inhibit growth Furthermore condensation may also build up on the tape so the adhesive needs to be strong and waterproof enough to thoroughly seal one well off from adjoining ones Q Do I need to set the gas flow prior to loading my plates into the HiGro HiGro Shaker User Manual ol A Yes Since it is impossible to know if the flow rate is very high when initially opening the cylinder and main shut off valves it is advisable to open these and set the gas flow prior to loading the sample plates If the flow rate is extremely high it is possible to blow liquid from one well into adjoining ones 6 When ending a run should I use the motion button to stop the shaking of HiGro without turning down the speed A NO Pressing the motion button causes the HiGro to stop shaking very abruptly from any speed Repeated use of the m
62. the insides of the chambers during the run they can damage the chambers and cause the HiGro to become out of balance 7 Replace the chamber lid and fasten the latches 6 6 BALANCE INDICATOR On the right side of the front panel of the HiGro is the Balance Indicator To prevent damage to the instrument or harm to users this safety feature shuts off the shaking motion if there is an incident which causes the HiGro to become unbalanced Under normal operation ie when the HiGro is balanced the indicator light will be off If during operation the HiGro becomes out of balance the light will come on and the HiGro will stop moving The HiGro can become unbalanced due to improper loading of the chambers by not evenly distributing plates or by not loading cassettes into all four chambers This indicator can also be activated if the HiGro is bumped or tilted during operation Once the Balance Indicator safety feature has been engaged the motion control button will still be activated and lighted and the speed read out will display the set point To properly shut down the HiGro turn the speed knob counter clockwise completely deactivate the motion control button and turn off the power to the HiGro If the problem that triggered the indicator is not apparent eg improper chamber loading then it is best to try to reproduce the problem Shut down the HiGro as described above and then restart the system If the Balance Indicator is triggered
63. the reg ulator box Whether it is better to increase or decrease the amount of air or oxygen will depend on the existing parame ters being used Please refer to Table 4 in the manual for suggested levels Keep in mind that with oxygenation less is usually better if there is uncertainty d If the wells are thick and cloudy in appearance yet the viable density and or the DNA yield is very low the samples are probably overgrowing and beginning to lyse To avoid this try reducing the growth time run length e If growth is poor in deep well plates it may be necessary to use 2mm soda glass beads 1 per well to improve aeration in the wells 9 2 MECHANICAL ELECTRICAL PROBLEMS Problem The lid latch on one of the chambers will not release because the adjacent chamber ts too close Suggestion s Depending on what position the HiGro stops in the inside latch on chamber 3 or 4 may be too close to chamber 1 or 2 respectively to release To solve this activate the motion button making sure that the speed knob is turned down completely and turn it up just slightly e g to Srpm Allow the HiGro to rotate until the chambers are further apart and the lid latch can be undone Problem Read out on temperature controller fluctuates while shaking or says Er 3 and or Er 4 or chamber does not heat up or heats up slowly Suggestion s Please call Customer Support at GeneMachines for assistance with these problems Telephone 650 508
64. traints for attaching the tank to a wall a wall Other Some air tanks have CGA 346 male tank Customers with oxygen ready system do have outlet connections Customers with this tank the option of switching to using air with their type will need an additional adapter to be HiGros This requires changing the nipple able to attach the regulator set up provided and bolt on the regulator set up provided by by GeneMachines This adapter can be pur GeneMachines These items can be purchased chased from GeneMachines It is not from GeneMachines The customer should included with the purchase of the HiGro follow the purity recommendations from this table when choosing an air tank Items with a are required specifications all others are recommendations A Please note Oxygen should NEVER be used on systems that have not been specified as oxygen ready Gases other than air and oxygen should NEVER be used with any HiGro system While GeneMachines does not anticipate any problems occuring from switching from oxygen to air use with the HiGro as described in Table 2 customers should be aware that there are inherent risks in changing the regulator set up and gas types Customers should be very careful to select the proper type and purity of gases and to check that any changes to the regulator set up are done properly GENEMACHINES DISCLAIMS ANY AND ALL LIABILITY FOR ISSUES OR PROB LEMS THAT MAY OCCUR DURING OR DUE TO THE MODIFICATION OF THE REGULAT
65. uld be connected from the regulator on the gas tank to the water bottle 2 The water bottle should be filled with 200ml of dH 0 The end of the tub ing in the bottle should extend into the water FIGURE 6 1 Gas fittings on a chamber 3 From the water bottle the tubing connects to the IN fitting on the back Fitting for panel of the machine input tubing 4 After entering the HiGro gas travels through the flowmeter and the gas on the rear of inlet valves and then flows to each of the chambers see Figures 6 3 and 6 the chamber 5 5 On the outside rear of each chamber are two fittings Gas flows in Output fitting through the upper fitting and into the manifold Please see Figure 6 8 for on the rear of a picture of the manifold The manifold is a long fixture gold colored that the chamber is attached along the back of the chamber Gas flows out of the manifold and over plates from regularly spaced holes in the manifold 6 The lower fitting on the outside rear of each chamber is for exhaust Inside the HiGro out of sight tubing leads from this lower fitting to emerge at the OUT fitting on the back of the HiGro see Figures 6 1 and 4 4 6 2 SETTING GAS FLOW TO CHAMBERS 1 Ensure that the gas tank cylinder FIGURE 6 2 Regulator set up valve and the main shut off valve are closed Output pressure set with N Tank 2 Make sure that the HiGro is not run he regulator knob 7 aa p 5 ak pressur
66. ve or below the set point HiGro Shaker User Manual 1 Suggestion s The temperature controllers on the HiGro are only capable of heating They do not cool the chambers When the chambers reach the set point temperatures the controllers stop heating They will only start heating again when the temperature falls below the set point Therefore during a run the chambers may fluctuate about the set temperature as the cells grow However the fluctuation should not exceed more than 2 C If it does try auto tuning the temperature controllers Please see 10 2 Auto tuning page 46 for instructions on auto tuning Problem Uneven or poor overall growth Suggestion s Unfortunately these are challenging problems to solve because they depend on many variables Here are some things to try a To even out growth try using a 50 50 mixture of 1x LB and 1x TB medias b If the poor growth is occuring with oxygenation try delaying the oxygen flow for 2 to 4 hours after starting arun This will give each well the chance to achieve the minimum number of cells required so that the oxygen does not inhibit growth See Setting gas flow to chambers on page 27 for information about how to set the delay c If the wells are still fairly clear after a run try increasing the shaking speed and or adjusting the amount of air or oxygen to the chambers The best and easiest way to increase decrease gas flow is to increase the on time interval on
67. wn for each cloning system represent the different flow rates when using four three two or one chambers for growth For example if gas is shut off to two out of four chambers the flowmeter should be set to 4 SLPM Chart last updated 04 25 2001 5 2 6 Total run time 1 For most growth applications in the HiGro the ideal run length will be between 14 and 18 hours 2 The specific run time will depend on the growth parameters When first using the HiGro GeneMachines recommends checking cell densities using a plate spectrophotometer at different end points This will help determine the optimal growth time and avoid overgrowth and cell lysis of cultures a commonly reported problem 5 2 7 Gas flow Depending on the model of HiGro customers can use air or oxygen TIP CELL LYSIS When cultures are overgrown or not well aerated cell lysis white filamentous 5 2 7 1 Air material will be evident in the wells Lack of aeration can be due to a too slow shaking speed or CO build up over the growth wells To improve aeration try increasing the shaking speed and or 1 In plasmid growth applications the main purpose of air flow is to keep CO from set using slightly more air or oxygenation Remember though too much oxygen tling above the wells This in turn helps opti will inhibit growth so a small increase in this type of gas is recommended mize aeration and prevent cell lysis Overgrowth is almost the opposite probl

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