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

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>Alan6017518
>Alan6017518
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*PCR of sequencing Library
*PCR of sequencing Library
*QPCR quantification
*QPCR quantification


===Fragmentation and end-polishing (Make blunt ends with 5'P)===
===Fragmentation and end-polishing (Make blunt ends with 5'P)===
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*End-Repair Reactions
*End-Repair Reactions
  {| {{table}}
  '''{| {{table}}
  | Fragmented DNA||85ul
  | Fragmented DNA||85ul
  |-
  |-
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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.
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*A-Tailing Reactions
*A-Tailing Reactions
{| {{table}}
'''{| {{table}}
| Blunet-end DNA||37ul
| Blunet-end DNA||37ul
|-
|-
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|  
|  
|}
|}
'''

Revision as of 18:03, 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 |- | |}