SPRI Purification of Sequencing Reactions
| Version Number: | 1.1 |
| Development Date: | 12-22-22 |
| Start R and d date: | 7-25-00 |
| Author: | Chris Detter, Damian Scott |
| Reviewed by: | Paul Richardson |
Summary
Precipitation of ET Terminator cycle sequenced products using an ethanol solution and SPRI beads
Materials & Reagents
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| Clear Plate Sealers | Edge BioSystems | 48461 |
| 96 Well Falcon Plates | Falcon | N/A |
| Stock Solutions | ||
| Etoh Precipitation Mix | JGI-see below | N/A |
| Formamide Loading Buffer | Amersham | US81095 |
| Reagents | ||
| Ethanol (100%) | AAPER | N/A |
| Water (100%) | Millipore Milli-Q system | N/A |
| Equipment | ||
| Plate Vortexer | VWR | N/A |
| Plate Shaker (Vibrax) | IKA | N/A |
| 96 well Plate Holder | Perkin Elmer | N/A |
| Multidrop 96 well | Titertek | N/A |
| Tecan Plate Washer | Tecan | N/A |
Procedure
Post Sequencing Reaction Cleanup/Precipitation
1. Retrieve sequencing plates from thermocyclers
2. Transfer 10ul seq rxn into 96 well Falcon plate using Hydra96
3. Add 7.5 ul of beads (diluted 1:5 in H20) per well
4. Add 68 ul of 100% ETOH to each well using Multidrop
5. Cover plate with clear seal
17. Shake plates on VWR plate vortexer
20. Vortex 2 minutes (setting = 6)
21. Let stand 10 min on bench top
22. Clear on magnet for ~ 2 min
23. Use Tecan plate washer to wash
24. Air dry ~ 20 min
25. Redissolve in 15ul Formamide
26. Shake 10 min on medium using Vibrax mixer
27. Clear on magnet for ~ 2 min
28. Transfer ~ 11ul to Robbins PCR plate while on magnet using Hydra96
29. Freeze (-20 C) until loaded on MegaBACE
Reagent/Stock Preparation
1. Wash Beads 3x in 10mM Tris
2. Dilute beads 1:5 in dH20
Instrument Settings
EtOH Multidrop
Column = 12 Volume = 68
Formamide Multidrop
Column = 12 Volume = 15
Tecan Plate Washer
Wash 3x with 70% ETOH in 200 ul total volume
Questions or Comments: Detter2@llnl.gov
