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.