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Kun:LabNotes/ASE/2009-7-20
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==Test the idea of doing SNP capturing directly on RNA== *Probes: CES8k18bp: 14ng/ul *Samples: **Hues6-ES ds-cDNA, 50ng/ul (2009-03-11) **Hues6-ES total RNA 781ng/ul A B ds-cDNA total RNA 50ng/ul 781ng/ul Sample 4ul 4ul CES8k18bp 2ul 2ul 10x buffer 2ul 2ul H2O 11ul 11ul 94C 2min -> 58C 16h -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul to A or MonsterScript RT 2U/ul to B, , AmpLigase 1U/ul) -> 58C 2h -> 94C 1min -> 37C 1min -> add 2ul Exo I & III (1ul RNase H to B) -> 37C 2h -> 4C hold. ===PCR=== PCR x 2 Template: 10ul 2X iProof MasterMix 50ul 100uM AmpF6.3rSol 0.4ul 100uM AmpR6.3rSol 0.4ul 50X SYBG I: 0.4ul H2O 38ul 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 15sec) x 8 -> (98C 10sec -> 72C 20sec) x 12 -> 72C 3min -> 4C hold. *The amplification curves came close to plateau at 12th cycles. The capturing seems to worked for both reactions. So I repeat the amplification on the remaining 10ul capturing reactions. *I got 200ul amplicons for each sample. I did E-gel SizeSelection on 120ul amplicons. It didn't go very well because of (i) high salt concentration in the PCR buffer; (ii) too much DNA. Nonetheless, the size-selected DNA were purified with Qiaquick columns together with the remaining 80ul amplicons. *I did a quantitative PAGE gel on all the four DNA samples. The bands are fuzzy due to the presence of ethanol in the eluted DNA. *Based on the PAGE gel, the estimated concentrations are: CES8k18bp-Hues6-DSCp: 4.5ng/ul = 38nM CES8k18bp-Hues6-tRp: 3.3ng/ul = 28nM ===Solexa sequencing=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Amount loaded''' | align="center" style="background:#f0f0f0;"|'''Library conc.''' |- | Lane 1||phiX||3pmole|| |- | Lane 2||phiX||4pmole|| |- | Lane 3||NA12878||4pmole||10nM |- | Lane 4||Hues6ES-DSp||5pmole||38nM |- | Lane 5||HUES6ES-tRp||5pmole||28nM |- | Lane 6||Hybrid1-EB||5pmole||606nM |- | Lane 7||Hybrid2-ES||5pmole||395nM |- | Lane 8||Hybrid2-EB||5pmole||303nM |- | |} *Lane 4 & 5 failed due to the use of a wrong primer. *Repeated the sequencing on 07/28/09, and obtained ~2M reads per lane. However, the majority of the reads either didn't match to the targets at all, or didn't cover the SNPs. I think there is something wrong with the libraries. The capturing reactions need to be repeated.
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