- Reamplify library and ship it out for shearing
'
|
'
|
'
|
x4
|
X1.1
|
Library |
12 |
0.25 |
1 |
1.1
|
2X Phusion |
50 |
50 |
200 |
220
|
AmpF6.3NH2 (10uM) |
2 |
2 |
8 |
8.8
|
AmpR6.3NH2 (10uM) |
2 |
2 |
8 |
8.8
|
10X SYBR Green I |
2 |
2 |
8 |
8.8
|
dH2O |
32 |
43.75 |
175 |
192.5
|
|
- 98C 1min->6X(98C 10 sec, 58C 20 sec, 72C 20sec)->72C 5 min
Construction of Solexa sequencing library from padlock captured PCR products using NEBNext DNA Sample Prep Master Mix Set 1 Kit (E6040S/L)[edit]
- Info Read the FAQ!!!
- Protocol
- The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II.
- For 60bp run, we can use the library size of >180bp
- For 80bp runs, the lower limit for the sequencing libraries should be 200bp.
- If the library size is too small, some of the 80bp reads will reach to the other end of the adaptor sequences. we don't want to waste the sequencing $$ on the regions we don't want
- The total size of the adaptors is 105bp, and there is another ~25bp of the capturing arm
- The upper limit should be ~50bp above the lower limit
Overview[edit]
1-7 PGP1,8 PGP1 & 9 PGP1[edit]
- Fragmentation and end-polishing
- Size Selection
- Ligation
- PCR of sequencing Library
- QPCR quantification
Fragmentation and end-polishing (Make blunt ends with 5'P)[edit]
- The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
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.
- 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 40 ul EB.
- Measure concentration with nanodrop
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 2009-11-16 15hr 00min.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 20ul EB.
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 |
18 |
18
|
10uM AmpR6.3Sol |
- |
18
|
10uM AmpF6.3Sol |
18 |
-
|
2X Phusion |
450 |
450
|
50X SYBR Green I |
3.6 |
3.6
|
H2O |
324 |
324
|
|
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
|
|
CVI-F
CVI-R
CVF-F
CVF-R
1-7 PGP1-iPS-F
1-7 PGP1-iPS-R
9 PGP1-iPS-F
9 PGP1-iPS-R
PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.
File:PCR of sequencing library.jpg
- TBE Gel verification (10well)
- 15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
- 15ul h2o+3ul 6X loading dye+2ul sampleFile:ZhangLab 2 2009-11-06 13hr 42min.jpg
- 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 2 lanes with 25bp ladder Mix in a .2ml tube(30ul H2O+9ul 6X loading dye+1ul 25bp ladder)/2
- Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells