AlanFung:LabNotes/Sequencing/2009-11-11
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Rescue CVF-N[edit]
- 20ul of CVF-N library, with buffer added instead of iProof master mix
Ethanol Precipitation[edit]
- Mix 50ul 100% ethanol, 2ul 3M NaoAC, and 1ul glycoblue
- Store at -80C for 20min
- Centrifuge 10,000 for 20min at 4C
- Discard supernatant
- Add in 750ul 75%ethanol
- Spin 10,000 rpm for 5 min at 4C
- Discard supernatant
- let dry in hood for 10 min
- Add in 21ul ddh2o
Repeat PCR of sequencing library with the following sets
Work on Set 8 DF, 8 FS, 9 DF, 9 FS, 8 PGP1-iPS[edit]
Size Selection[edit]
- 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.
File:ZhangLab 2 2009-11-11 14hr 28min.jpg File:ZhangLab 2 2009-11-11 14hr 30min.jpg
- 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.[edit]
- 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[edit]
- Include CVF-N set
- Total 6 sets of libraries
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 to 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.
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.