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