Construction of CV-iB libraries with the USER/S1 protocol[edit]
PCR with dUTP[edit]
- 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[edit]
DNA 60ul
USER 6ul
37C 1 hour
S1 Nuclease digestion[edit]
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)[edit]
- 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.
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[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 2009-12-01 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.[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 23ul EB.
Nanodrop
A,C: 15.9ng/ul*23ul=365.7ng
B,D,E: 15.6ng/ul*23ul=358.8ng
PCR of sequencing library[edit]
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
|
|
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[edit]
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 |
|
|
Dilute to 10nM |
Sample (ul) |
ddH2O
|
CV-iB1 |
5.000 |
45.595
|
CV-iF1 |
5.000 |
37.428
|
CV-F1 |
5.000 |
48.852
|
CV-iB (A,C) |
5.000 |
93.765
|
CV-iB (B,D,E) |
5.000 |
92.811
|
PGP8A |
5.000 |
3.652
|
|