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