Kun:LabNotes/ASE/2009-7-20
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Test the idea of doing SNP capturing directly on RNA[edit]
- 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[edit]
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[edit]
' | Sample | Amount loaded | 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.