AlanFung:LabNotes/Capturing/BisFirstExon 2010 1 11
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QPCR Validation for CV-iPS B & F[edit]
8ul h2o+1.5ul 6x loading dye+0.5ul 25bp ladder 6.5ul h2o+1.5ul 6X loading dye+2ul sample Run at 250V for 20min File:ZhangLab 2 2010-01-11 10hr 54min.jpg
Capturing for CV-iPS B, CV-iPS F, NA12878, NA18507[edit]
CV-iPS B-17.5ng/ul CV-iPS F-20.4ng/ul NA12878-17.6ng/ul NA18507-12.4ng/ul
Capturing System | CV-iPS-B | CV-iPS-F | NA12878 | NA18506 |
Volume of probes | 7.993 | 7.993 | 7.993 | 7.993 |
Volume of templates | 11.429 | 9.804 | 11.364 | 16.129 |
Volume of H20 | 4.079 | 5.703 | 4.144 | 0.000 |
Volume of 10X Ampligase buffer | 1.500 | 1.500 | 1.500 | 1.500 |
Total Volume | 25.000 | 25.000 | 25.000 | 25.000 |
Capturing System Setup[edit]
95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 6h ->
6 cycles (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold Saved as biscap
Total Volume of SLN Mix Needed (uL) | 7 | ||
Stock Stoffel Concentration (U/uL) | 10 | Stoffel Concentration Needed (U/uL) | 2 |
Stock Ampligase Concentration (U/uL) | 5 | Stock Ampligase Concentration Needed (U/uL) | 0.5 |
Stock DNTP (mM) | 10 | DNTP Needed (mM) | 1 |
10X Ligase Buffer | 10 | ||
H20 | |||
Volume of H2O | 3.5 | ||
Volume of DNTP | 0.7 | ||
Volume of Stoffel | 1.4 | ||
Volume of Ampligase | 0.7 | ||
Volume of Ligase Buffer | 0.7 | ||
Total Volume of SLN Mix (uL) | 7 | ||