AlanFung:Protocol/Construction of Solexa sequencing Library: Difference between revisions
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*[[Media:DNASPMMS1.pdf|Protocol]] | *[[Media:DNASPMMS1.pdf|Protocol]] | ||
*The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II. | *The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II. | ||
*For 60bp run, we can use the library size of >180bp | |||
*For 80bp runs, the lower limit for the sequencing libraries should be 200bp. | |||
*If the library size is too small, some of the 80bp reads will reach to the other end of the adaptor sequences. we don't want to waste the sequencing $$ on the regions we don't want | |||
*The total size of the adaptors is 105bp, and there is another ~25bp of the capturing arm | |||
*The upper limit should be ~50bp above the lower limit | |||
=Overview= | =Overview= |
Revision as of 08:35, 30 October 2009
Construction of Solexa sequencing library from padlock captured PCR products using NEBNext DNA Sample Prep Master Mix Set 1 Kit (E6040S/L)
- Info Read the FAQ!!!
- Protocol
- The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II.
- For 60bp run, we can use the library size of >180bp
- For 80bp runs, the lower limit for the sequencing libraries should be 200bp.
- If the library size is too small, some of the 80bp reads will reach to the other end of the adaptor sequences. we don't want to waste the sequencing $$ on the regions we don't want
- The total size of the adaptors is 105bp, and there is another ~25bp of the capturing arm
- The upper limit should be ~50bp above the lower limit
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 |