Direct End Sequencing of BAC Clones
| Version Number: | 1 |
| Start R and D Date: | 6-1-00 |
| Author: | Damian Scott |
| Reviewed by: | Paul Richardson |
Summary
Direct end sequencing of BAC clones.
Materials & Reagents
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| Clear Plate Sealers | Edge BioSystems | 48461 |
| Cycle Sequence Sealers | Robbins | N/A |
| Stock Solutions | ||
| Etoh Precipitation Mix | JGI-see below | N/A |
| Formamide Loading Buffer | Amersham | US81095 |
| ET Terminator Kit | Amersham | N/A |
| pBAC R primer | IDT | N/A |
| Reagents | ||
| Ethanol (100%) | AAPER | N/A |
| 7.5M Ammonium Acetate | Amersham (comes with ET kit) | N/A |
| Equipment | ||
| Plate Vortexer | VWR | N/A |
| Eppendorf 5810 Centrifuge | Eppendorf | N/A |
| 96 well Plate Holder | Perkin Elmer | N/A |
| Stir Bar | VRW | N/A |
| PE 9700 Thermocycler | Perkin Elmer | N/A |
Procedure
Cycle Sequencing (20ul rxn)
1. Remove ET kit from freezer and unfreeze on ice
2. Remove BAC DNA from refrigerator and place on ice
3. Remove 250pmol/ul stock pBAC R primer from freezer and thaw on benchtop
4. Using Hydra 96, add 12 ul of BAC DNA to each well of a 96 well Robbins plate
5. Using a Matrix pipettor, add 8ul of ET kit with primer to each well of a 96 well Robbins plate
6. Centrifuge up to 1000rpm and back down
7. Place spun down Robbins plate in thermocycler
Post Sequencing Reaction Cleanup/Precipitation
1. Prepare ETOH precipitation solution
2. Add 57ul/well of ETOH precipitation to cycle sequenced Robbins plate
3. Vortex on medium setting for 30 seconds
4. Centrifuge for 60 minutes @4000rpm
5. Remove plate and turn upside down to remove ETOH
6. Spin upside down on a paper towel for 1 minute @ 500rpm
7. Air dry in dark drawer for 15 minutes
8. Add 10ul/well of formamide
9. Vortex on medium setting for 30 seconds
10. Spin up to 1000rpm and back down
11. Store at -20 C
Reagent/Stock Preparation
ET Terminator Kit
1. Unfreeze ET kit and immediately place of ice
2. Uunfreeze 250pmol/ul stock primer solution, vortex on high setting for 15 seconds, add 4ul of primer per 1ml of ET kit
3. Mix by inverting several times.
ETOH Precipitation Preparation
1. Add 55ml of 100%ETOH to a 250 ml beaker
2. Add 2ml of 7.5M Ammonium Acetate into same beaker
3. Using a stir bar, mix solution for 2 minutes with a plate stirrer
Instrument Settings
Thermocyclers Settings
95C 4min HOLD
95C(15sec)
50C (10sec) 40cycles
60C (2min)
Hold at 4C
Questions or Comments: Scott50@llnl.gov
