Kun:LabNotes/ASE/2009-7-20: Difference between revisions
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m (→PCR) |
>Jie deng |
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Line 7: | Line 7: | ||
ds-cDNA total RNA | ds-cDNA total RNA | ||
50ng/ul 781ng/ul | 50ng/ul 781ng/ul | ||
Sample | Sample 4ul 4ul | ||
CES8k18bp 2ul 2ul | CES8k18bp 2ul 2ul | ||
10x buffer 2ul 2ul | 10x buffer 2ul 2ul | ||
H2O | H2O 11ul 11ul | ||
94C 2min -> 58C 16h | 94C 2min -> 58C 16h | ||
Line 23: | Line 23: | ||
50X SYBG I: 0.4ul | 50X SYBG I: 0.4ul | ||
H2O 38ul | H2O 38ul | ||
98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 15sec) x 8 -> (98C 10sec -> 72C 20sec) x | 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 15sec) x 8 -> (98C 10sec -> 72C 20sec) x 12 -> 72C 3min -> 4C hold. |
Revision as of 22:25, 21 July 2009
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.