AlanFung:Protocol/Genome Analyzer: Difference between revisions
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==Incorporation Mix== | ==Incorporation Mix== | ||
'''Required Materials''' | '''Required Materials''' | ||
''' | |||
'''*The volumes of the dNTPs in the various kits are specific to that kit''' | |||
*Do not mix and match dNTP vials form different cycle number kits | '''*Do not mix and match dNTP vials form different cycle number kits''' | ||
''' | |||
*dNTPs: ff-dATP, ff-dCTP, ff-dGTP, ff-dTTP | *dNTPs: ff-dATP, ff-dCTP, ff-dGTP, ff-dTTP | ||
2 each for the 36-cycle kits | 2 each for the 36-cycle kits |
Revision as of 18:36, 25 June 2009
Preparing Reagents for the Genome Analyzer
Genome analyzer Reagent Compartment & Reagent Position
- Position of each reagent in the Genome Analyzer
NOTE:LABEL ALL REAGENT CONTAINERS WITH THE APPROPRIATE NUMBER Position Reagent Volume Container 1 Incorporation Mix Varies w/ cycle # 50ml amber tube 2 DI H2O 250ml bottle 3 Scanning Mix 50ml 50ml tube 4 High Salt Buffer 250ml bottle 5 Incorporation buffer 150ml 250ml bottle 6 Cleavage Mix 50ml 50ml tube 7 Cleavage Buffer 70ml 250ml bottle
Scanning Mix
Required Materials *Scanning Reagent *Scanning Buffer *Filter Unit *Scanning Mix Tube
- Add the Scanning Reagent vial to the Scanning Buffer on ice
- Rinse the empty vial into the Scanning Buffer three times with 1 ml Scanning Buffer
- Invert the Scanning Buffer several times to mix
- Leave on ice for 10 minutes
- Invert several times to mix. Check to ensure that the solids are completely dissolved
- Filter the Scanning Mix with the provided Scanning Mix Filter Unit
- Pour 50 ml of the Scanning Mix into the 50 ml Scanning Mix Tube
*If you are running more than 26 cycles on the Genome Analyzer, you must top off the Scanning Mix at the end of the first day of the sequencing run. The tube should be approximately one-third empty after running for six or seven hours
- Record the weight of the reagent in the lab tracking worksheet
- Leave the remaining solution in the Filter Unit at 4C for later cycles
- Keep the Scanning Mix on ice until ready to load
Incorporation Mix
Required Materials
*The volumes of the dNTPs in the various kits are specific to that kit *Do not mix and match dNTP vials form different cycle number kits *dNTPs: ff-dATP, ff-dCTP, ff-dGTP, ff-dTTP 2 each for the 36-cycle kits 1 each for the 18-cycle and 26-cycle kits *10X Incorporation Buffer v2.0 (5 ml) *dNTP Mix Diluent *SBS Polymerase 2 for the 36-cycle kits 1 for the 18-cycle and 26-cycle kits *Magnesium *Plastic Syringe *0.22 μm Cellulose Acetate Syringe Filter *50 ml Amber Tube