USER MANUAL: g-TUBE
Contents
1. a a Si 1 6kbp 14500 rpm 4 a 2 8 kbp 10000 rpm E a 3 10 kbp 8000 mm 2 a 4 20 kbp 6500 ram 1 1s 1 i os os F ai pare Uppermarier om y Biounaiaer eee T T Figure 3 150 ul of Lambda was fragmented with g TUBEs following the different settings of table 1 Results are from a 0 5 agarose gel top insert and from a 12k Agilent chip run on a 2100 Bioanalyser lower insert RPM values are for an Eppendorf MiniSpin plus centrifuge Part Number O10154RevC 9 Page Date June 20122. is free from particulate matter and the DNA is fully dissolved If in doubt please see 5 in the FAQ section 3 Close the g TUBE a Screw the cap on very firmly Please see 11 in the FAQ section if you are unable to screw the cap on very firmly by hand b Proceed to the next step within 15 minutes Part Number 010154 Rev C 2IPage Date June 2012 ___Covaris 4 Load the g TUBEs screw cap up into the centrifuge If necessary use an extra g TUBE to balance the rotor a Set the centrifuge to the speed specified in Table 1 Spin the samples for the Processing time listed in Table 1 b Remove the g TUBEs from the centrifuge visually check to determine if the entire sample has drained from the upper chamber of the g TUBE if not see 6 in FAQ section Part Number 010154 Rev C 3 Page Date June 2012 Eppendorf 5424 and 5415 R centrifuges Speed RPM rgeted size 6 kbp Bkbp 10 kbp 20 kbp Mass of DNA aug 11 000 7 200 6 000 4 200 Bue 11 000 7 200 6 000 4800 15 pg 13 200 8 600 7 200 5 800 30 ng 13 200 10 000 8 600 7 200 Processing time seconds 30sec 60sec 60 sec 60sec Eppendorf MiniSpin plus Speed RPM Targeted size 6 kbp Bkbp 10 kbp 20 kbp Mass of DNA ape 14 500 9 400 8 000 5 500 Bue 14 500 3 400 8 000 6 300 15 pg gt 11 200 9 400 7 600 30 pg 13 000 11 200 3 400 Processing time seconds 30 sec 60 sec 60sec 60sec Tabl
3. threads The sample isn t lost and is recoverable First pipette out the sample in the cap then close the tube and spin it screw cap down at low RPM for a few seconds You can now pipette the remaining sample from the cap My DNA isn t fully in solution or I have impurities in my sample Incubate the DNA in a microcentrifuge tube for 20min at 35C vortex frequently and then centrifuge your sample at 14 000 RCF for 15min to pellet DNA that isn t in solution You can also filter the sample using a small filtration device with 0 45 um membrane for example 4mm Millex filter from Millipore After the first spin I can still see some sample in the upper chamber what should 1 do Impurities in the sample may have clogged the g TUBE We advise to keep repeating the 1 spin step screw cap up until all the sample goes through Then you can invert the tube screw cap down and spin the sample using the normal process If the sample will not pass into the second chamber then the ruby is blocked with sample debris Try the following procedure o Flip the g TUBE screw cap down and spin it at 4300 RCF for 30seconds Part Number 010154 Rev C 6lPage Date June 2012 10 11 Recover the sample from the cap following the usual procedure o Filter the sample using a small filtration device with 0 45 um membrane for example 4 mm Millex filter from Millipore Use a NEW g TUBE and follow the normal protocol Fm getting DNA fr
4. USER MANUAL g TUBE INTRODUCTION The g TUBE is a single use consumable designed to shear genomic DNA into large fragments with a mean ranging from 6 kbp to 20 kbp g TUBE works with a bench top centrifuge and the final size of the DNA fragments is controlled by the acceleration rate and speed of the centrifuge USER SUPPLIED MATERIALS Genomic DNA o Mass of DNA between 4ug and 30ug per g TUBE Buffer DI water or TE or 10 mM Tris Cl pH 8 5 o Non degraded starting size larger than 45 kbp DNA fully in solution vortexed for 10s and pre warmed at room temperature 20 C to 30 C No particulate matter in the sample o Eppendorf MiniSpin plus microcentrifuge Eppendorf pn 022620207 for 110V version See 1 in FAQ for others compatible centrifuges SUPPLIES Part Number g TUBE 10 520079 g TUBE Case 100 520104 g TUBE Prep Station 10 500291 eppendortsis a registered trademark Part Number 010154 Rev C 1lPage Date June 2012 Covaris The objective is to have the sample pass two times through an orifice at a controlled rate The first spin is to transfer the sample to the bottom of the tube The second spin is to transfer the sample from the bottom of the tube and into the cap PROTOCOL 1 Place the g TUBE in the LOAD position of the stand screw cap up 2 Remove the screw cap from the g TUBE and load 150ul of sample into the top of the tube a Please ensure that your sample
5. agments smaller than expected Check the quality of your starting material by running it on a gel Degraded genomic DNA appearing as a smear on a gel or a starting material smaller than 45kbp will result in smaller DNA fragments than high quality genomic DNA It may be possible to obtain the fragment size that you require by adjusting the settings speed lower centrifuge Do I need to use wide bore pipette tips to transfer DNA No you can use regular pipette tips What is the operating temperature of the g TUBEs Please operate your centrifuge between 20 C and 30 What is the storage temperature of the g TUBEs Before use store your g TUBEs between 20 C and 30 C My finger tight isn t strong enough to properly tighten the cap on the g TUBE You can use the stand as a tool to tighten the cap Hold the g TUBE vertical with the screw cap up while using the stand as a wrench to tighten the cap Figure 1 Using the stand as a wrench to tighten the cap Part Number 10154RevC 7 Page Date June 2012 Covaris SUPPLEMENTARY DATA Reproducible Ladder kop cre 15 os tinn Ladder kbp 10 8 5 5 H 3 os Figure 2 150ul of Lambda DNA 50ug ml was fragmented to 10kbp using g TUBEs 18 different replicates are presented Parameters were those in table 1 Part Number Date 010154 Rev June 2012 BiPage Versatile 123 Ladder kbp i Ladder kop w lt
6. e 1 Use these tables to determine the centrifuge speed RPM for the amount of DNA and the targeted DNA fragments size These combinations are not available with Eppendorf MiniSpin plus centrifuge Acceler n should never exceed 16000g or RCF Part Number 010154 Rev C alpa Date June 2012 5 Invert the g TUBEs place them in the rotor screw cap down and spin them aga the same speed and duration a Proceed to the next step within 15 minutes 6 Transfer the g TUBE from the centrifuge to the UNLOAD position of the stand keeping the screw cap down all the time 7 Unscrew the tube body leaving the screw cap containing the sample in the stand Recover the sample from the screw cap You may tilt the stand to aid sample recovery Part Number 10154RevC 5 Page Date June 2012 Covaris Trouble shooting Frequently Asked Questions FAQ Which centrifuges are compatible Eppendorf MiniSpin plus Eppendorf Model 5415 R with temperature set at 30 C Eppendorf Model 5424 What is the range of sample volumes g TUBE is validated for use with 150pl sample Other sample volumes will affect the result Is it possible to store sample in a g TUBE No the sample has to be processed witi the g TUBE immediately after processing 15 minutes after loading and removed from Ididn t recover my entire sample what happened Ifthe g TUBE screw cap isn t tightly closed some sample may move into the screw
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