AlanFung:LabNotes/Sequencing/2009-12-1
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Construction of CV-iB libraries with the USER/S1 protocol
PCR with dUTP
- Mix CV-iB #A and #C at 3:1 ratio. 10ul A + 1ul C.
- Mix CV-iB #B, #D, #E at 2:1:1 ratio. 4ul B + 4ul D + 3ul E.
x2 DNA 10ul 2X Taq Master mix: 200ul 1mM dUTP: 8ul 100uM AmpF6.3NH2: 0.8ul 100uM AmpR6.3NH2: 0.8ul H2O 181ul 94C 2min -> 8x (94C 30sec -> 60C 30sec -> 72C 30sec) -> 72C 3min. Purify each amplicon with two Qiaquick columns. Elute w/ 32ul EB buffer in each tube.
USER digestion
DNA 60ul USER 6ul 37C 1 hour
S1 Nuclease digestion
S1 Digestion | |
10X S1 Nuclease Buffer | 8 |
DNA after USER Digestion | 66 |
S1 Nuclease (10U/ul) | 2 |
ddH2O | 4 |
- 37C 10mins
- Minelute purification. Elute w/16uH2O x2 for each set
- Nanodrop
A,C:20.7ng/ul*32ul=662.4ng B,D,E:32.1ng/ul*32ul=1027.2ng
- Fragmentation and end-polishing
- Size Selection
- Ligation
- PCR of sequencing Library
- QPCR quantification
Fragmentation and end-polishing (Make blunt ends with 5'P)
- Make up to 85ul with dh2o (53ul each)
- 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 40ul EB buffer.
Nanodrop A,C: 12.1ng/ul*40ul=484ng B,D,E: 19.1ng/ul*40ul=764ng
- 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 22 ul EB.
- Measure concentration with nanodrop
A,C: 14.4ng/ul*22ul=316.8ng B,D,E: 24.7ng/ul*22ul=543.4ng
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 2009-12-01 12hr 31min.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.
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 23ul EB.
Nanodrop A,C: 15.9ng/ul*23ul=365.7ng B,D,E: 15.6ng/ul*23ul=358.8ng
PCR of sequencing library
Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC
AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA
- prepare 2 master mix tube
' | F | R |
10uM Solexa_PCR_up | 4.4 | 4.4 |
10uM AmpR6.3Sol | 0 | 4.4 |
10uM AmpF6.3Sol | 4.4 | 0 |
2X Phusion | 110 | 110 |
50X SYBR Green I | 0.88 | 0.88 |
H2O | 79.2 | 79.2 |
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 |
- 90ul mix per well
- Setup
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)
- 10ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
- 10ul h2o+3ul 6X loading dye+2ul sample
File:ZhangLab 2 2009-12-01 15hr 00min.jpg File:ZhangLab 2 2009-12-01 15hr 27min.jpg
- Mix the amplicons with two sets of primers
- Purified with Minelute columns, elute with 13ul EB buffer
QPCR Quantification
Setup Stock Phi-X (1nM) CV-iB1 (1/10) CV-F1 (1/10) CV-iB-B,D,E (1/10) Stock Phi-X (1nM) CV-iB1 (1/10) CV-F1 (1/10) CV-iB-B,D,E (1/10) Stock Phi-X (1nM) CV-iB1 (1/10) CV-F1 (1/10) CV-iB-B,D,E (1/10) Stock Phi-X (1nM) CV-iB1 (1/10) CV-F1 (1/10) CV-iB-B,D,E (1/10) Amp Phi-X (1nM) CV-iF1 (1/10) CV-iB-A,C (1/10) PGP8A Amp Phi-X (1nM) CV-iF1 (1/10) CV-iB-A,C (1/10) PGP8A Amp Phi-X (1nM) CV-iF1 (1/10) CV-iB-A,C (1/10) PGP8A Amp Phi-X (1nM) CV-iF1 (1/10) CV-iB-A,C (1/10) PGP8A
X16 | X1.1 | ||
DNA | 2.000 | ||
Syb_FP5 (100uM) | 0.200 | 3.200 | 3.520 |
Syb_RP7 (100uM) | 0.200 | 3.200 | 3.520 |
2X Phusion | 25.000 | 400.000 | 440.000 |
50X SYBR Green I | 0.200 | 3.200 | 3.520 |
H20 | 24.000 | 384.000 | 422.400 |
872.960 | |||
- Dilute sample, 1ul in 9ul ddh2o
- mix 2ul 1/10 sample with 98ul mix
- aliquot 24ul to each well
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min
Result | ||||
PhiX | 10.749 | Cycle Difference | 2^ | x10 (nM) |
CV-iB1 | 7.410 | 3.339 | 10.119 | 101.190 |
CV-iF1 | 7.664 | 3.085 | 8.486 | 84.855 |
CV-F1 | 7.320 | 3.429 | 10.770 | 107.704 |
CV-iB (A,C) | 6.445 | 4.304 | 19.753 | 197.530 |
CV-iB (B,D,E) | 6.459 | 4.290 | 19.562 | 195.622 |
PGP8A | 6.636 | 4.113 | 17.304 | |