Alice:LabNotes/2009-12-5
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Solexa sequencing library preparetion
Fragmentation and end-polishing (Make blunt ends with 5'P)
- 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.
- 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
CViB: 54ng/ul
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.
File:ZhangLab 2 2009-12-06 14hr 17min.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.
- 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.
End-repaired & size selected DNA | 36ul |
40uM adaptor2 | 2ul |
5X Quick Ligase Buffer | 10ul |
Quick Ligase | 2ul |
- Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB.
PCR of sequencing library
Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC
AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA
- prepare 2 master mix tube
Ligation products | 10 | 10 |
10uM solexa PCR up | 2 | 2 |
10uM AmpR6.3Sol | 2 | - |
10uM AmpF6.3Sol | - | 2 |
2X Phusion | 50 | 50 |
50X SYBR Green I | 0.4 | 0.4 |
H2O | 36 | 36 |
PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.
- TBE Gel verification (10well)
- Mix the amplicons with two sets of primers
- Purified with Minelute columns, elute with 32ul EB buffer
TBE Gel Size Selection
- Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel
- Load 1 lane with 25bp ladder (20ul H2O+20ul 6X loading dye+1ul 25bp ladder)/2
- Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells