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

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 14: Line 14:
*End-Repair Reactions
*End-Repair Reactions
   {| {{table}}
   {| {{table}}
   | Fragmented DNA||85ul
   | Fragmented DNA||   85ul
   |-
   |-
   | 10X End Repair Bufer||10ul
   | 10X End Repair Bufer||   10ul
   |-
   |-
   | End Repair Enzyme Mix||5ul
   | End Repair Enzyme Mix||   5ul
   |-
   |-
   |  
   |  

Revision as of 18:04, 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 (Make blunt ends with 5'P)

  • 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.
  • NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
  • A-Tailing Reactions
Blunet-end DNA 37ul
10X dA-Tailing Reaction Buffer 5ul
Klenow Fragment (3'-5' exo-) 3ul
H2O 5ul