Sephacryl Purification of Sequencing Reactions
| Version Number: | 1 |
| Development Date: | X |
| Start RND date: | X |
| Author: | Damian Scott |
| Reviewed by: | Paul Richardson |
Summary
This protocol is used as an alternative to the traditional methods of precipitating cycle sequencing reactions. This protocol uses a sephacryl plate to purify the cycle sequence product through centrifugation. The eluted product is run directly on the MegaBACE 3700.
Materials & Reagents
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| AutoSeq 96 Prototype Plate | Amersham | 27534096 |
| Cycleplate-96 ET Thin Wall PCR Plate | Robbins | 1055-90-3 |
| Cycle sequenced 96 or 384 well plate | PSF-sequencing group | N/A |
| Clear Plate Sealers | Edge BioSystems | 48461 |
| 96 well plate holder | Perkin Elmer | N/A |
| Tape | Scotch | N/A |
| Equipment | ||
| 96 well Hydra (100ul) | Robbins Scientific | N/A |
| Eppendorf 5810 Centrifuge | Eppendorf | 5810 |
Procedure
Preparation of Sephacryl Plate
1.Take AutoSeq 96 Prototype Plate and position on top of a 96 well Robbins Plate in 96 well plate holder. Tape in place.
2.Place the assembled plate(s) from step 1 into Eppendorf 5810 centrifuge. Centrifuge at 910 g for 5 minutes.
3.Remove plate(s) from centrifuge. Remove AutoSeq 96 Prototype Plate from the 96 well Robbins plate in 96 well plate holder. Discard 96 well Robbins plate that contains the waste from the AutoSeq 96 Prototype Plate.
4. Take AutoSeq 96 Prototype Plate and position on top of a labeled 96 well Robbins Plate in 96 well plate holder. Tape in place.
Addition of Sequenced Products
1.Remove cycle sequenced 96 well Robbins plate from thermocycler and place into a 96 well plate holder.
2. Using 96 well Hydra Program 4, fill from 96 well Robbins plate in 96 well plate holder.
3. Aliquot onto the AutoSeq 96 Prototype Plate positioned on top of a labeled 96 well Robbins Plate in 96 well plate holder.
4.Use wash function on Hyrda between each new plate.
Elution of Products
1.Take the AutoSeq 96 Prototype Plate positioned on top of a labeled 96 well Robbins Plate in 96 well plate holder with the aliquoted cycle sequenced products.
2. Place the assembled plate(s) from step 1 into Eppendorf 5810 centrifuge. Centrifuge at 910 g for 5 minutes.
3. Remove plate(s) from centrifuge. Remove AutoSeq 96 Prototype Plate from the 96 from the Robbins plate in 96 well plate holder.
4.Seal the 96 well Robbins plate with a clear plate sealer.
5.Turn in to MegaBACE group.
Questions or Comments: Scott50@llnl.gov
