Alice:LabNotes/2009-10-9
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iPS library construction
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
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
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
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
- Mix 20ul A_adaptor_b (100uM), 20ul A_adaptor_t (100uM), 5ul 10x Taq buffer, 55ul H2O.
- 94C 3min -> ramp to 20C at 0.1C/sec -> 4C hold.
- 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
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)