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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