AlanFung:Protocol/Construction of Solexa sequencing Library: Difference between revisions
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Mix the amplicons with two sets of primers, purified with Qiaquick columns. Typically, the PCR products are clean enough for sequencing without further size selection. | Mix the amplicons with two sets of primers, purified with Qiaquick columns. Typically, the PCR products are clean enough for sequencing without further size selection. | ||
==TBE Gel Size Selection== | |||
* Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel | |||
* Load 2 lanes with 25bp ladder Mix in a .2ml tube(30ul H2O+9ul 6X loading dye+1ul 25bp ladder)/2 | |||
* Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells |
Revision as of 23:02, 12 November 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 |
Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 40 ul EB.
- Measure concentration with nanodrop
Size selection using Invitrogen 2% SizeSelect gel
- Fill any unused well with 30ul EB Buffer
- Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube
- Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
- Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well
- Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
- Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
- Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.
- Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).
Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.
- Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
ul | |
End-repaired & size selected DNA | 36 |
40uM adaptor2 | 2 |
5X Quick Ligase Buffer | 10 |
Quick Ligase | 2 |
- Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 22ul EB.
PCR of sequencing library
Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC
AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA
Ligation products | 10 | 10 |
10uM solexa PCR up | 2 | 2 |
10uM AmpR6.3Sol | 2 | - |
10uM AmpF6.3Sol | - | 2 |
2X iProof master mix (Bio-Rad) | 50 | 50 |
50X SYBR Green I | 0.4 | 0.4 |
H2O | 36 | 36 |
- Setup 2 master mix
F | R | |
Ligation products | ||
10uM solexa PCR up | 13.2 | 13.2 |
10uM AmpR6.3Sol | 13.2 | - |
10uM AmpF6.3Sol | - | 13.2 |
2X iProof master mix (Bio-Rad) | 330 | 330 |
50X SYBR Green I | 2.64 | 2.64 |
H2O | 237.6 | 237.6 |
PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. File:20091112 125359.jpg
- TBE Gel verification (10well)
- 15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
- 15ul h2o+3ul 6X loading dye+2ul sample
File:ZhangLab 2 2009-11-12 14hr 46min.jpg File:ZhangLab 2 2009-11-12 14hr 48min.jpg Mix the amplicons with two sets of primers, purified with Qiaquick columns. Typically, the PCR products are clean enough for sequencing without further size selection.
TBE Gel Size Selection
* Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel * Load 2 lanes with 25bp ladder Mix in a .2ml tube(30ul H2O+9ul 6X loading dye+1ul 25bp ladder)/2 * Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells