Solexa sequencing library preparetion[edit]
Fragmentation and end-polishing (Make blunt ends with 5'P)[edit]
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.
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[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 2009-12-06 14hr 17min.jpg
- Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).
Ligation[edit]
- 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[edit]
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[edit]
- 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
File:ZhangLab 2 2009-12-07 21hr 35min.jpg