Revision as of 00:13, 13 January 2010 by >Alan6017518
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 |
|
|
|