Alice:LabNotes/2009-10-9: Difference between revisions
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===Ligation=== | ===Ligation=== | ||
*Assuming we have 100ng of 80bp fragments, and the Adaptor:Insert ratio is 50:1, we need ~100pmole of the adaptors. | *Assuming we have 100ng of 80bp fragments, and the Adaptor:Insert ratio is 50:1, we need ~100pmole of the adaptors. | ||
*Include a positive control. | *Include a positive control. | ||
Line 104: | Line 103: | ||
98C 30sec -> 11 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) | 98C 30sec -> 11 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) | ||
gel size selection: | gel size selection: | ||
[[Image:ZhangLab_2 2009-10-11 15hr 37min.jpg]] | [[Image:ZhangLab_2 2009-10-11 15hr 37min.jpg]] [[Image:ZhangLab_2 2009-10-11 15hr 38min.jpg]] | ||
[[Image:ZhangLab_2 2009-10-11 16hr 05min.jpg]] | [[Image:ZhangLab_2 2009-10-11 16hr 05min.jpg]] | ||
===QPCR quantification=== | |||
Template 1ul | |||
2x iProof mix 25ul | |||
Solexa_PCR_up (10uM) 0.2ul | |||
Solexa_PCR_lo_PE (10uM) 0.2ul | |||
H2O 24ul | |||
50X SYBG I 0.2ul | |||
98C 30sec -> 20 cycles of (98C 10sec -> 62C 20sec -> 72C 15 sec) -> 72C 3min |
Latest revision as of 00:55, 20 October 2009
iPS library construction[edit]
The five libraries were pooled together as the following ratio (second PCR-amplification was not performed, these are amplified capture product) pooled all the subsets together using the ratio: 1-100%(Jan09 1+2+3); 2-50%(Jan09 4+5DS); 3-73%(Jan09 6+7); 4-73%(Mar09 2+3, Jan09 5SS)
pooled all the subsets together using the ratio: 1-100%; 2-50%; 3-73%; 4-72% DF1 DF2 DF3 DF4 concnetration(ng/ul): 32.9ng/ul 14.7ng/ul 41.4ng/ul 12.9ng/ul volume needed (ul): 4ul 4.5ul 2.3ul 7.5ul -------------------------------------------------------------------------- total (ng): 132ng 66ng 96ng 96ng FS1 FS2 DF3 DF4 concnetration(ng/ul): 28ng/ul 13.8ng/ul 26.1ng/ul 9.4ng/ul volume needed (ul): 7ul 7ul 5.4ul 5.6ul -------------------------------------------------------------------------- total (ng): 86.1ng 43ng 63ng 63ng ips 1 ips 2 ips 3 ips 4 concnetration(ng/ul): 21.7ng/ul 9.7ng/ul 20.2ng/ul 7.9ng/ul volume needed (ul): 6ul 6.5ul 4.5ul 10ul -------------------------------------------------------------------------- total (ng): 130.2ng 65.1ng 95ng 95ng CV-ips 1 CV-ips 2 CV-ips 3 CV-ips 4 concnetration(ng/ul): 31.3ng/ul 10.2ng/ul 25ng/ul 12ng/ul volume needed (ul): 4ul 6.1ul 3.7ul 7ul -------------------------------------------------------------------------- total (ng): 125.2ng 62.6ng 91.4ng 91.4ng CV-fb 1 CV-fb 2 CV-fb 3 CV-fb 4 concnetration(ng/ul): 28ng/ul 13.8ng/ul 26.1ng/ul 9.4ng/ul volume needed (ul): 4ul 4ul 3.1ul 8ul -------------------------------------------------------------------------- total (ng): 112ng 56ng 82ng 81ng
Qiaquick column purification
MmeI digestion[edit]
DNA 23ul 10x NEB buffer 4 4ul 1mM SAM 6ul 2U/ul MmeI 8ul
MinElute purification and elute in 100ul EB.
- Sent out the five libraries to Harvard for Covaris shearing
- Received five sheared samples from Billy, ~400ng in ~80ul each
- Use the NEBNext DNA Sample Prep Master Mix Set I kit for library construction.
- Use the Y-adaptor for ligation.
End repair[edit]
DNA ~50ul End Repair 10X buffer 10ul End Repair Enzyme Mix 5ul H2O ~30ul Total 100ul Room temperature for 20min, purified with Qiaquick column, eluted with 39ul EB
A Tailing[edit]
End repaired DNA 37ul dA-Tailing buffer (10x) 5ul Klenow exo- 3ul H2O 5ul Incubate at 37C for 30min
- Purify with Qiaquick columns, eluted with 30ul EB.
- Perform size selection on 20ul DNA with 2% size selection gel, extract the ~100bp bands.
Ligation[edit]
- Assuming we have 100ng of 80bp fragments, and the Adaptor:Insert ratio is 50:1, we need ~100pmole of the adaptors.
- Include a positive control.
A-tailed DNA 15ul 5X QuickLigase buffer 10ul 20uM Adaptor mix 5ul Quick T4 ligase 5ul H2O 15ul RT for 15min Purified with MinElute columns, eluted with 15ul EB.
PCR[edit]
Check the size distribution of the PCR products with 1ul of ligation products first Template 1ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 0.4ul Solexa_PCR_lo_PE (10uM) 0.4ul H2O 49ul 50X SYBG I 0.4ul 98C 30sec -> 13 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) File:ZhangLab 2 2009-10-09 15hr 40min.jpg
Repeat the PCR with real samples Template 5ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 0.4ul Solexa_PCR_lo_PE (10uM) 0.4ul H2O 45ul 50X SYBG I 0.4ul 98C 30sec -> 11 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) gel size selection: File:ZhangLab 2 2009-10-11 15hr 37min.jpg File:ZhangLab 2 2009-10-11 15hr 38min.jpg File:ZhangLab 2 2009-10-11 16hr 05min.jpg
QPCR quantification[edit]
Template 1ul 2x iProof mix 25ul Solexa_PCR_up (10uM) 0.2ul Solexa_PCR_lo_PE (10uM) 0.2ul H2O 24ul 50X SYBG I 0.2ul 98C 30sec -> 20 cycles of (98C 10sec -> 62C 20sec -> 72C 15 sec) -> 72C 3min