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

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*Incubate tubes at RT for 30minutes.
*Incubate tubes at RT for 30minutes.
*Perform a Qiaquick purification and elute with 39ul EB buffer.
*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
{| {{table}}
| Blunet-end DNA||37ul
|-
| 10X dA-Tailing Reaction Buffer||5ul
|-
| Klenow Fragment (3\'-5\' exo-)||3ul
|-
| H2O||5ul
|-
|
|}

Revision as of 18:01, 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