マニュアル GeneGel HyRes Starter Kit
Contents
1. GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA GE Healthcare Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany GE Healthcare Bio Sciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA www gehealthcare com lifesciences GE imagination at work2. Pipette about 0 5 ml of insulating fluid kerosene onto the center of the cooling plate of the GenePhor Electrophoresis Unit Use tissue paper to spread it evenly over the surface Using the grid on the cooling plate as a guide position the gel in the center of the cooling plate with the sample wells at the cathode side Fig 4 Avoid trapping air bubbles under the gel 4 To prevent drying apply the electrode wicks as soon as possible Fig 5 Wetting electrode wicks with electrode buffer electrophoresis Preparing the electrode wicks Place two stacks of four standard electrode wicks each on any hard flat non absorbent surface such as a glass plate or dish 9 Use a pipette to apply 6 ml of the appropriate electrode buffer to each stack of electrode wicks Fig 5 Use GeneGel HyRes Native Electrode Buffer with native gels Use GeneGel HyRes Denaturing Electrode Buffer with denaturing gels After thoroughly wetting the electrode wicks stroke them with the tip of a bent forceps to distribute the buffer evenly and remove air bubbles Blot the electrode wicks on a paper towel to remove excess buffer Q Place one stack of electrode wicks on the gel near the cathodic end of the GenePhor cooling platform and place the other stack of wicks on the gel at the anodic end epi GeneGel Hyres Starter Kit electrophoresis Important Wipe the platinum electrode wires b
3. dration buffer Fig 2 For even buffer distribution carefully lift the opposite side of the film am 80 6452 59 e p4 Preparing gels These instructions can be used to rehydrate either H yR es N ative or HyRes Denaturing gels Be sure to use the appro priate rehydration buffer Rehydrating native gels Use GeneGel H yRes N ative Rehydration Buffer as supplied to rehydrate native gels Rehydrating denaturing gels Place 8 4 g urea 7 M in a beaker and add 13 5 ml GeneG el H yRes Denaturing Rehydration Buffer M ix thoroughly Use this solution with GeneGel H yR es Denaturing gels 0 Pipette 20 ml of rehydration buffer into the rehydration tray 9 Open the gel package by cutting along two sides of the package Remove the gel from the package and remove the plastic covering sheet covering the gel Set one edge of the film with the gel surface downward into gel rehydration buffer Fig 1 Slowly lower the rest of the gel film into the buffer Take care to avoid trapping air bubbles 4 To distribute the buffer evenly lift the other side of the film using forceps and lower it once more into the buffer again taking care to avoid trapping air bubbles under the gel Fig 2 Note The HyRes Native gel is made of a proprietary poly acrylamide matrix It is slightly thicker and more brittle than the HyRes Denaturing gel Important The gel surface must be free of droplets before beginnin
4. 5 ml total volume 4 94 ml Native Gel Rehydration Buffer 8 ul xylene cyanol solution 1 50 pl EDTA solution 0 1 M Denaturing solution 25 ml total volume Combine 23 75 ml formamide 0 625 ml 1 xylene cyanol solution 0 625 ml 1 bromophenol blue solution May be stored up to 12 months at 4 8 C Note To avoid overloading the recommended total amount of DNA in a 4 ul sample volume should not exceed 0 2 ug Note To avoid overloading the recommended total amount of DNA in a 4 ul sample volume should be between 0 5 ug and 1 pg preparing samples and standards Preparing samples for native gels If necessary dilute the sample with sample buffer see page 2 2 For size standard dilute the GE Healthcare 100 Base pair ladder code no 27 4007 01 The recommended load is 150 200 ng per well Preparing samples for denaturing gels To dilute samples and standards If necessary dilute the sample with denaturing solution see page 2 9 For size standard dilute the GE Healthcare 100 Base pair ladder code no 27 4007 01 The recommended load is 150 200 ng per well Mix the samples thoroughly and heat them 3 5 min to 95 C Chill samples in an ice water bath before applying to the gel e p3 GeneGel Hyres Starter Kit preparing gels Important Avoid excessive folding or bending of GeneGel HyRes gels Fig 1 Lower the film with the gel surface downward into the rehy
5. GeneGel HyRes gels remove the acrylic buffer strip positioner from the GenePhor Electrophoresis Unit during the run to prevent condensation on the gel Note Total running time for denaturing gels should not exceed 90 minutes electrophoresis Running conditions Native gels Table 1 gives recommended settings and running conditions for use with GeneGel HyResNative Gels Table 1 Running conditions HyRes Native Electrode Buffer max max max temperature voltage current watts time Phasel 12 C 180 V 22 mA 5 W 40 min Phase 2 12 C 390 V 26 mA 11 W 75 min For 40 200 bp fragments set Phase 2 to 75 min At the end of this time the bromophenol blue front reaches the anode For 100 300 bp fragments set Phase 2 to 130 min At the end of this time the xylene cyanol front reaches the anode For 200 400 bp fragments set Phase 2 to 200 min At the end of this time the xylene cyanol front is projected to be 1 cm past the anode Denaturing gels Table 2 gives recommended settings and running conditions for use with GeneGel H yRes Denaturing Gels During sample loading set the temperature to 20 C Table 2 Running conditions with HyRes Denaturing Electrode Buffer max max max temperature voltage current watts time Phase 1 40 C 100 V 10 mA 5 W 10 min After Phase 1 cover the gel with a sheet of polyester film Phase 2 50 C 300 V 12 mA 10 W 45 min After 55 min electrophoresis the bromo
6. 6 manual DNA fragment analysis GeneGel HyRes Starter Kit for high resolution electrophoretic DNA typing 218 234 bp 5 i i if 3 ERAS i 9 Sa E E R fone TE a LI 4 4 jis NCR o e a RS Z e um 80 6452 59 Rev B0 04 02 GE imagination at work Page finder Introduction Rena ii Preparing samples and standards 2 Preparing solutions w w ata Xe aa te ai te ok fa tak ta tak Te T 2 Preparing samples for native gels 3 Preparing samples for denaturing gels 3 Preparing Gels 4 2 4 4 Electrophoresis 0 6 Preparing the electrode wicks o o o 7 Loading the samples 8 RUNNING conditions cours 9 Detection iride 10 Product information 0 ooooo ooo 11 epi GeneGel Hyres Starter Kit introduction am 80 6452 59 e pii Introduction The GeneGel H yR es Starter Kit provides materials for the highest resolution DNA separation on the GenePhor electrophoresis unit The kit is suitable for a variety of elec trophoretic analyses including variable number tandem repeats VN TR low complexity differential display reverse transcription DDRT and most short tandem repeat STR analysis With a separation range of 40 250 bp the GeneGel H yRes Denaturing gels provide 2 4 bp resolution in as little as 2 5 h total separation and detection time GeneGel H yRes Native gels require 4 h total separation and det
7. Native Gels 80 6451 26 GeneGel HyRes Denaturing Gels 80 6451 45 GeneGel HyRes Native Buffer Kit 80 6451 64 Includes Rehydration buffer Electrode buffer electrode wicks 40 drying boards 5 GeneGel HyRes Denaturing Buffer Kit 80 6451 83 Includes Rehydration buffer Electrode buffer electrode wicks 40 drying boards 5 polyester film GeneGel Standard Electrode Wicks 80 6452 02 GenePhor Electrophoresis unit 18 1115 82 Related products EPS 1001 Power Supply 18 1130 03 Hoefer Processor Plus base unit Includes pump reagent tubing protocol key 80 6444 04 Gel Staining Tray Pack 19 x 29 cm 80 6444 80 Includes tray support PTFE coated staining tray lid magnets gasket level and Gel Staining Protocol Guide PlusOne DNA Silver Staining Kit 17 6000 30 100 Base pair Ladder 27 4007 01 1 ug ul in TE buffer 100 ug e pll Notes am 80 6452 59 e p12 GenePhor Hoefer and PlusOne are trademarks of GE Healthcare Bio Sciences Ltd GE Healthcare is a trade mark of General Electric Company AII goods and services are sold subject to the terms and condi tions of sale of the company within the General Electric Company group that supplies them A copy of these terms and conditions is available on request Printed in the USA GE Healthcare UK Ltd GE Healthcare Place Little Chalfont Buckinghamshire England HP7 9NA GE Healthcare Bio Sciences AB SE 751 84 Uppsala Sweden
8. ection time The GeneGel H yR es Starter Kit contains pre cast dry gels for both native and denaturing applications The native gels offer high resolution of ds DNA fragments The denaturing gels are comparable to a sequencing gel format for the sepa ration of ss DNA fragments Rehydrate each gel with ts respective Rehydration Buffer and soak the included electrode wicks in the corresponding Electrode Buffer The combination of the electrode and gel rehydration buffers form discontinuous buffer systems designed for efficient DN A separations Use the GeneGel H yR es Starter Kit with the GenePhor Electrophoresis Unit Following separation we recommend using the H oefer Processor Plus together with the PlusO ne DNA Silver Staining Kit for high sensitivity gel staining The plastic backing on all GeneGel gels allows the stained gel to be easily handled and dried for accurate evaluation and permanent documentation of results Warning For research use only Not recommended or intended for the diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Handling Storage Store the buffers at ambient temperature Store the gels at 4 8 C Expiry Please refer to the kit packaging components Components Gels 3 GeneGel HyRes Native gel 0 65 mm after rehydration 2 GeneGel HyRes Denaturing gel 0 43 mm after rehydration Buffers GeneGel HyRes Native Rehydration Buffer wit
9. efore and after each electrophoresis run with wet tissue paper Important Do not use the buffer strip positioner with GeneGel HyRes gels See page 6 Fig 6 Loading samples with a pipette am 80 6452 59 e p8 Loading the samples Prepare the samples according to the directions on page 3 Use a pipette to load the recommended 4 ul of sample or size standard into each well Fig 6 9 Lower the electrode lid onto the electrode wicks Verify that the electrodes come in even contact with the stacks of electrode wicks Lower the safety lid until it clicks shut Connect the power supply Set the power supply to the recommended values Table 1 Table 2 or Table 3 and press Start Check the current reading to verify power is properly connected milliamps greater than 0 6 If using a denaturing gel at the end of phase 1 switch off the power supply Open the safety lid and the electrode holder plate Place the polyester film over the gel surface between the two stacks of electrode wicks to prevent gel drying Close the electrode holder plate and the safety lid Continue with phase 2 Important When using the GeneGel HyRes gels remove the acrylic buffer strip positioner from the GenePhor Electrophoresis Unit during the run to prevent condensation on the gel Note Determine the optimal run time based on the size of the fragments of interest Important When using the
10. g electrophoresis Fig 3 Dry the surface of the rehy drated gel with the edges of a gel drying board preparing gels 6 To assure even rehydration and prevent the gel from sticking to the chamber place the rehydration tray on a rotating shaker for 90 minutes at a vigorous rotation rate 80 rpm During agitation the gel should bump against the walls of the rehydration tray After 90 minutes the gel will have swelled to a final thickness gt 0 5 mm 6 After rehydration remove the gel from the rehydration tray and place it gel side up on filter paper Q To remove excess buffer gently place the gel on the benchtop Use the edge of a gel drying board to wipe buffer off the gel surface Fig 3 until a squeaking sound is produced from the wiping of the drying board against the gel surface approximately 2 3 wipes The gel is now ready to use for electrophoresis e p5 GeneGel Hyres Starter Kit electrophoresis Important When using the GeneGel HyRes gels remove the acrylic buffer strip positioner from the GenePhor Electrophoresis Unit during the run to prevent condensation on the gel Note Wear gloves when handling the gels to avoid contamination particularly when using sensitive silver stains Fig 4 Positioning the gel with sample wells at cathode am 80 6452 59 e p6 Electrophoresis 0 Switch on the GenePhor Electrophoresis Unit and set to the recommended temperature 9
11. h bromophenol blue 75 ml GeneGel HyRes Denaturing Rehydration Buffer 45 ml GeneGel HyRes Native Electrode Buffer 40 ml GeneGel HyRes Denaturing Electrode Buffer 30 ml Electrode Wicks 40 GeneGel Standard Electrode Wicks 5 Gel drying boards Other materials required not supplied with this kit Formamide code 17 1320 01 Xylene cyanol dye Bromophenol blue code no 17 1329 01 100 Base pair Ladder recommended code 27 4007 01 Kerosene Urea for GeneGel HyRes Denaturing gel Forceps Rotating shaker platform Rehydration tray code 18 1117 60 epi GeneGel Hyres Starter Kit preparing samples and standards Note The maximum loading volume of the wells in GeneGel HyRes gels is 4 pl am 80 6452 59 e p2 Preparing samples and standards The sensitivity of the detection method you plan to use determines the minimum amount of sample that can be used When possible samples should be diluted with sample buffer to yield 6 ng of each DNA fragment present in a 4 ul loading volume Use this as a guideline when using silver staining for detection Preparing solutions Prepare the following solutions for use in diluting samples and standards 0 1 M EDTA 1 0 g EDTA disodium salt Dissolve and make up to 25 ml with distilled water 1 w v xylene cyanol or bromophenol blue solution 0 1 g solid xylene cyanol or bromophenol blue Dissolve and make up to 10 ml with distilled water Sample buffer for native gels
12. phenol blue and 70 base fragments reach the anode Xylene cyanol runs with 100 base fragments e p9 GeneGel Hyres Starter Kit detection Important Immediately cover GeneGel HyRes Native gels after air drying for 1 hour These gels become brittle and shatter with excessive drying am 80 6452 59 e p10 Detection For reproducible and sensitive silver staining use the Hoefer Processor Plus with gel staining trays and the PlusO ne DNA Silver Staining Kit Because of the special matrix of the GeneGel H yRes N ative gels and the high concentration of urea used with the GeneG el H yRes Denaturing gels we recommend the modifications shown in Table 3 After air drying the stained gels cover them with polyester film and store at room temperature Table 3 Modified Silver Staining of GeneGel HyRes Gels step time min solution 1 Fixing 40 175 ml 0 6 Benzene sulphonic acid in 24 v v ethanol 2 Wash 3x10 175 ml 0 06 Benzene sulphonic acid dilute 5X Fix 1 50 in distilled H 0 3 Silver solution 40 175 ml 0 2 w v AgNO3 0 07 benzenesulphonic acid 4 Wash 2 5 Developing 7 175 ml distilled H O 175 ml 2 5 Na2CO3 175 ul 37 formaldehyde 175 ul 2 sodium thiosulfate 6 Stop preserve 20 175 ml 1 0 v v Acetic acid solution 5 sodium acetate 10 glycerol product information Product information product code no GeneGel HyRes Starter Kit 80 6451 07 GeneGel HyRes
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