User manual / Technical Information
Contents
1. Operating Manua Capillary Running Module MINI 2811 1 amp MAXI 2816 1 WARNING Please read the entire operator s manual thoroughly before operating this unit Warning Like all apparatus run by electricity these units are capable of delivering potentially lethal voltage when connected to a power supply They should be operated only by qualified technically trained personnel The capillary electrophoresis modules from ROTH are designed for long term laboratory use and to obtain reproducible results Please spend a few moments reading the instruction manual thoroughly These units comply with the statutory CE safety rules 73 23 EEC Low voltage directive IEC 1010 1 1990 plus amendment 1 1992 EN 61010 1 1993 BS EN 61010 1 1993 Please verify that you received the unit completely and without any damage Any faults or losses have to be reported to ROTH immediately ROTH can not accept responsibility for goods that were sent back without informing them Please retain all packaging material until the warranty period has expired For further information please contact us at Tel 0721 5606 0 SPECIFICATIONS Technical features Durable acrylic construction All acrylic joints chemically bonded Doubly insulated cables rated safe up to 1 000 volts Gold plated electrical connectors corrosion free and rated safe up to 1 000 volts Recessed power connectors integral wit2. 1 cm of the top of the capillary 5 Overlay the top of the gel solution with water saturated isobutanol The 1 cm gap will serve as a loading chamber for the sample Allow the gel solution to set for at least 30 min D 2 Capillary Method 1 If capillary action is the preferred pouring method prepare double the amount of acrylamide solution to that in the recipe above Pipette 70 of the acrylamide solution into a flat bottomed beaker and stand the capillary tubes upright in the acrylamide solution Allow the tubes to equilibrate with the acrylamide solution Check the height of the acrylamide in the tubes If the tubes are full so that there is less than a 1 cm nonfilled space at the top remove some of the acrylamide solution from the beaker until the height is 1 cm from the top If there is a greater thanicm space at the top add more acrylamide solution so that the solution rises in the tubes until there is a 1cm space at the top Fill the 1 cm space which will act as the sample well with water saturated isobutanol 2 Leave to polymerise which will normally take 1 2 hours 3 After polymerisation remove the water saturated isobutanol 4 The tube gels can now be removed from the beaker and inserted into the slots in the Internal Running Module with the 1 cm sample well facing upwards Alternatively they can be stored wrapped in a damp paper towel and Clingfilm at 4 C The tubes may contain a residue of acrylamide on the outside and
3. Blue over the tube gel allowing it to set Electrophorese as usual for slab gels until the tracker dye has advanced the required distance down the gel The samples can be visualized using any of the standard staining methods or can be blotted H Further reagents Ampholytes pH Conductivity pH lower pH upper Art range mS cm limit limit No Roti lyte pH 3 5 5834 Roti lyte pH 3 6 5835 Roti lyte pH 3 7 5839 Roti lyte pH 3 10 5841 Roti lyte pH 4 6 5845 Roti lyte pH 4 7 5850 Rotilyte pH 5 6 5851 Roti lyte pH 5 7 5856 Roti lyte pH 5 8 5857 Roti lyte pH 6 7 5858 Roti lytepH6 8 6 8 0 15 0 55 6 240 5 8 240 6 5860 Roti lyte pH 7 9 5861 Roti lyte pH 8 10 8 10 0 1 0 3 7 5 0 5 9 5 0 6 5864 Acetone gt 99 5 for synthesis 5025 Ammonium persulfate APS 9592 Bromophenol Blue A512 DTT 6908 Filter papers e g CL67 Glycerol 3783 or 7530 Isobutanol 6772 Levelling table N854 NaOH 32 T196 Phosphoric acid 6366 Roti Green fluorescent staining solution 1000 Roti Load 1 6 x reducing K929 Roti Load 2 6 x non reducing K930 Roti Red fluorescent staining solution 1045 Rotiphorese 10 x SDS PAGE running buffer 3060 Rotiphorese Blue A152 Rotiphorese Gel 40 37 5 1 T802 Roti Stock 20 SDS Solution ready to use 1057 SDS 2326 SDS Pellets CN30 Silver staining Roti Black P Protein L533 Silver staining Bleaching Roti Bleach N766 TEMED 2367 Tris AE15
4. Tris Cl prebuffered 9090 Triton X 100 3051 Urea 2317 Water double distilled 3478 Xylene cyanol A513 Carl Roth GmbH Co KG SchoemperlenstraBe 3 5 76185 Karlsruhe Postfach 100121 76231 Karlsruhe Telefon 49 721 5606 0 Telefax 49 721 5606 149 E Mail info carlroth de Internet www carlroth de s s 11 2014 For further data and safety recommendations please see our catalogue or the internet at carlroth de or carlroth com
5. afety lid firmly making sure that the electrical connectors form a good contact The current will gradually decline as the pH gradient forms After 30 min stop the power supply turn off the mains supply and disconnect the power leads Load 2 to 10 ul of sample previously prepared in sample buffer see above with a syringe into the top of each gel The sample should sink to the bottom of the well Perform the separation step at 400 V for the time period suggested in the table 2 depending on the length of the capillary gel Meanwhile prepare a 12 5 polyacrylamide resolving gel using a preparatory comb with one large slot see Info brochure Polyacrylamide Gels technical tipps and general considerations A gradient gel can be used to resolve proteins of highly contrasting molecular weights F At the End of the Run 1 Q N Perform the End of Run stage as suggested in table 1 Turn the power supply settings to zero turn off the mains supply and disconnect the power leads Turn off the water supply if the unit is being cooled Remove the safety lid by pressing against the tank from the top and take out the lid by the handles Remove the capillary module from the tank and carefully remove the capillaries Use the nylon thread to draw the gel gently out of the capillary Let it slide onto the gel extraction platform and cut off the thread Alternatively Using 24ga needle gently squirt water into the top of the glass
6. capillary so that the gel is extruded onto the gel extraction platform For 2 dimension runs see G 2 D Size Determination Phase Tubes can be cleaned using a mild detergent and rinsing in distilled water A clean sheet of foam rubber placed at the bottom of the sink serves as a tube support and minimises the risk of tube damage Empty the tank buffer chambers with a vacuum line and trap or carefully decant the buffer away from the electrical connectors Rinse the chambers with distilled water then dry the electrode connectors with tissue Ensure that the connectors are clean and dry before usage or storage 2 D Size Determination Phase 2 dimension gel Equilibrate the capillary gel for 30 mins in the running buffer to be used for 2 dimension For 2 dimension we recommend to use 1 5 mm gels Remove the gel from the buffer solution and align the gel extraction platform with the top of the SDS PAGE Allow the gel to slide gently onto the top of the SDS PAGE gel making sure that it is in contact with the resolving gel over its entire length The gel should be casted using a blank or 2 D comb For details on the casting of slab gels and the use of the Roth Vertical Electrophoresis Units see the Info brochure Polyacrylamide Gels technical tipps and general considerations and the manual accompanying the vertical electrophoresis chambers Load the desired protein markers for analysis Pipette molten 0 5 agarose with 0 04 Bromophenol
7. e tubes DONOT use abrasive creams or scourers NEVER allow organic solvents or chromic acid to come into contact with the plastic components e Handle clean tubes with gloved hands remove any fingerprints with acetone IMPORTANT Lubricate the Capillary Tubes and Internal Running Module slots with distilled water or running buffer before inserting the tubes D Pouring of Capillary Gels Ampholyte Gel Solution 7 8 ml distilled water 1 2ml 40 ampholyte solution of the pH range required see H Further reagents 2 52 ml Rotiphorese Gel 40 37 5 1 13 119 Urea to prepare 9 M solution 2 43 ml 20 Triton X 100 15ul TEMED 150 ul 10 v v Ammonium Persulphate D 1 Sealing Method 1 Insert a thin nylon thread inside of the capillary Be sure that it protrudes on every side for several centimeters 2 Seal one end of each glass capillary with Parafilm and place upright in the running module Insert the glass capillary from the bottom upwards to prevent the Parafilm getting dislodged 3 Prepare the following gel solution using the ampholyte corresponding to the desired pH gradient There is sufficient volume for twenty 8 cm or ten 17 cm capillary gels 4 Filla 10 ml syringe attached to a 24ga needle with gel solution Insert the needle into the glass capillary until itis 1 cm from the bottom Fill the capillary with gel solution slowly withdrawing the needle as the fluid level rises Stop once the gel solution rises to within
8. ear protective clothing when working with acrylamide and follow and observe the working instructions directions for disposal carefully Polymerized gels contain residue of unpolymerized monomer Please wear always protective gloves while working Caution During electrophoresis very low quantities of various gases are produced at the electrodes The type of gas produced depends on the composition of the buffer employed To disperse these gases make sure that the apparatus is run in a well ventilated area B General Care and Maintenance Clean the apparatus with destilled water only Important Acrylic plastic is not resistant to aromatic or halogenated hydrocarbons ketones esters alcohols over 25 and acids over 25 they will cause crazing especially of the UV transparent plastic and should not be used for cleaning Do not use abrasive creams or scourers Dry components with clean tissues prior to use e g ROTH tissues ref 0087 1 The replacement platinum electrodes are partially shrouded for protection However when cleaning the main tank do not use cleaning brushes in the electrode area Usually a thorough rinse with distilled water is all that is required Ensure that the connectors are clean and dry before usage or storage C Capillary Tube Preparation e Clean the outside of the tubes in a mild laboratory detergent A piece of wire with a cotton wool plug placed over the end can be used to clean the insides of th
9. h the safety lid 0 2 mm diameter platinum electrodes 99 99 pure User replacable platinum electrodes VLAN NNAN AN Environmental Conditions This apparatus is intended for indoor use only The unit can be operated safely at an altitude of 2000 m The normal operating temperature range is between 4 C and 65 C Maximum relative humidity 80 for temperatures up to 31 C decreasing linearly to 50 relative humidity at 40 C ANNAN All Roth products available for delivery have undergone rigorous quality controls AVAILABLE ACCESSORIES All accessories can be purchased from Carl Roth GmbH Co KG Please use the indicated ordering numbers Tank without lid Y005 1 Cooled tank without lid Y006 1 Replacement lid for tank Y009 1 Running module Y007 1 Casting module Y008 1 U shaped fixing plate seals for running module 8 per set Y010 1 Seals for running module 8 per set YO11 1 Gaskets for casting module 2 per set T796 1 Replacement screws for running module 4 per set Y0O12 1 Replacement platin electrode 0 2 mm T794 1 Blanking Ports silicone 10 pieces 2815 1 Glass capillaries MINI 8 cm length 1 mm internal diameter 10 pieces 2812 1 Glass capillaries MINI 8 cm length 4 mm internal diameter 10 pieces 2813 1 Glass capillaries MAXI 17 cm length 1 mm internal diameter 10 pieces 2817 1 Glass capillaries MAXI 17 cm length 4 mm internal diameter 10 pieces 2818 1 Gel extraction platform MINI for max 8 cm gel
10. may need cleaning with distilled water before insertion E 1st Dimension IEF Phase Buffer and run conditions will vary according to the type of ampholyte used The following conditions are given as guidelines only and apply when a pH gradient of approx 4 8 is the ampholyte used Other ampholytes will require different buffer solutions Please consult your laboratory manuals Sample Overlay Solution 4 8g urea 0 5 ml 40 ampholyte solution of the pH range required distilled water to 40 ml Sample Buffer 5 4g urea 1ml 40 ampholyte solution of the pH range required 2ml 20 Triton X 100 0 1g dithiothreitol DTT distilled water to 10ml Fill the tank with low pH anode solution 0 1 phosphoric acid according to amounts given in table 2 The buffer has to reach the white plastic ridge since the lower electrode is located here Insert the blanking ports into the grommets if using fewer than 10 capillary gels in the running module Remove the Parafilm from the bottom of each glass capillary and insert the capillary running module into the tank Fill the inner chamber with high pH cathode solution 20 mM NaOH see table 2 ensuring that the top of each capillary is immersed completely Add 5 ul of sample overlay solution see above to the top of each gel before replacing the lid Perform the prerun pre focusing step for 30 min at 200 V see table 2 The prerun stage is recommended as it helps set up the pH gradient Replace the s
11. s 1 piece 2814 1 Gel extraction platform MAXI for max 17 cm gels 1 piece 2819 1 Tab 1 Operational Unit Max voltage V Max current per gel mA a 1000 V 110 mA MAXI ir Tab 2 Technical Specifications Units MINI MAXI Capillary Inner Outer Length Inner Outer Length Dimensions Diameter Diameter Diameter Diameter 1 mm 5 mm 80 mm 1 mm 5 mm 170 mm Recommended Upper Buffer Chamber Gel Tank Upper Buffer Chamber Gel Tank Buffer Volume 100 ml 600 ml 200 ml 1 600 ml Recommended Prerun Separation End of Run Prerun Separation End of Run Running Conditions 200 V 400 V 500 V 200 V 400 V 500 V for IEF or 30 min 3h 30 min 30 min 6 18h 30 min Capillary Gels 100 Vh 1 200 Vh 250 Vh 100 Vh 2 400 Vh 250 Vh USING THE CAPILLARY GEL ELECTROPHORESIS UNITS A Safety Precautions Please read the entire instruction manual thoroughly before using the apparatus Please also follow instructions of the tanks electrophoresis unit Always isolate electrophoresis units from their power supply before removing the safety cover Isolate the power supply from the mains first then disconnect the leads Do not exceed the maximum operating voltage or current see table 1 Do not operate the capillary electrophoresis units in metal trays Do not fill the unit with running buffer above the maximum fill lines Do not move the unit when it is running Acrylamide is a volatile concentrated neurotoxin which is suspected to be carcinogenic Please always w
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