AlanFung:Protocol/Construction of Solexa sequencing Library: Difference between revisions

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*QPCR quantification
*QPCR quantification


==Fragmentation and end-polishing
==Fragmentation and end-polishing==
*The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
*The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
*End-Repair Reactions
*End-Repair Reactions
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*Incubate tubes at RT for 30minutes.
*Perform a Qiaquick purification and elute with 39ul EB buffer.

Revision as of 17:57, 29 October 2009

Construction of Solexa sequencing library from padlock captured PCR products using NEBNext DNA Sample Prep Master Mix Set 1 Kit (E6040S/L)

Overview

  • Fragmentation and end-polishing
  • Size Selection
  • Ligation
  • PCR of sequencing Library
  • QPCR quantification

Fragmentation and end-polishing

  • The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
  • End-Repair Reactions
Fragmented DNA 85ul
10X End Repair Bufer 10ul
End Repair Enzyme Mix 5ul
  • Incubate tubes at RT for 30minutes.
  • Perform a Qiaquick purification and elute with 39ul EB buffer.