AlanFung:LabNotes/Sequencing/2009-12-1: Difference between revisions
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>Alan6017518 (New page: ==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 +...) |
>Alan6017518 |
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(23 intermediate revisions by the same user not shown) | |||
Line 22: | Line 22: | ||
| S1 Digestion|| | | S1 Digestion|| | ||
|- | |- | ||
| 10X S1 Nuclease Buffer|| | | 10X S1 Nuclease Buffer||8 | ||
|- | |- | ||
| DNA after USER Digestion|| | | DNA after USER Digestion||66 | ||
|- | |- | ||
| S1 Nuclease (10U/ul)|| | | S1 Nuclease (10U/ul)||2 | ||
|- | |- | ||
| ddH2O|| | | ddH2O||4 | ||
|- | |- | ||
| | | | ||
Line 34: | Line 34: | ||
*37C 10mins | *37C 10mins | ||
*Minelute purification. Elute w/16uH2O | *Minelute purification. Elute w/16uH2O x2 for each set | ||
=== | *Nanodrop | ||
== | A,C:20.7ng/ul*32ul=662.4ng | ||
===Ligation=== | B,D,E:32.1ng/ul*32ul=1027.2ng | ||
=== | |||
===PCR=== | *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 | |||
{| {{table}} | |||
| 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 | |||
{| {{table}} | |||
| 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. | |||
[[Image: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. | |||
{| {{table}} | |||
| ||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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''F''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| 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 | |||
[[Image:ZhangLab_2 2009-12-01 15hr 00min.jpg]] | |||
[[Image: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 | |||
{| {{table}} | |||
| ||||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 | |||
{| {{table}} | |||
| 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|| | |||
|- | |||
| | |||
|} | |||
{| {{table}} | |||
| 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 | |||
|- | |||
| | |||
|} |
Latest revision as of 03:16, 2 December 2009
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.
- 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[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 |
- 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[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 |