AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 64: | Line 64: | ||
*Aliquot 32ul to each well +8ul sample =40ul Total | *Aliquot 32ul to each well +8ul sample =40ul Total | ||
Perform PCR reaction in real-time thermocycler | Perform PCR reaction in real-time thermocycler | ||
{| {{table}} | {| {{table}} | ||
| | | NA12878||NA12878||NA12878||Jurkat (mix) | ||
|- | |- | ||
| Jurkat ( | | NA18507||NA18507||NA18507||Jurkat (mix) | ||
|- | |- | ||
| Jurkat (mix) | | NA20431||NA20431||NA20431||Jurkat (mix) | ||
|- | |- | ||
| | | CV-Fibr||CV-Fibr||CV-Fibr|| | ||
|- | |- | ||
| | | CV-iPS-B||CV-iPS-B||CV-iPS-B|| | ||
|- | |- | ||
| | | CV-iPS-F||CV-iPS-F||CV-iPS-F|| | ||
|- | |- | ||
| | | Jurkat||Jurkat||Jurkat|| | ||
|- | |- | ||
| | | Jurkat (old)||Jurkat (old)||Jurkat (old)|| | ||
| | |||
| | |||
|- | |- | ||
| | | | ||
|} | |} | ||
Step1 96C, 3m | |||
Step2 95C, 30s | |||
Step3 62C, 1m | |||
Step4 72C, 1m | |||
Step5 Go to step2 repeat 39 times | |||
Step6 72C, 5m | |||
Step7 4C, Forever |
Revision as of 19:23, 4 December 2009
qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)
- Start with 10ng template for each set of primer (30ng Total)
Sample | Sample Concentration ng/ul | To get 30ng | 1/10 Dilution | To get 30ng |
NA12878 | 17.6 | 1.7 | ||
NA18507 | 12.4 | 2.42 | ||
NA20431 | 50.6 | 0.59 | ||
CV-Fibr | 4.84 | 6.2 | ||
CV-iPS-B | 7.27 | 4.13 | ||
CV-iPS-F | 6.91 | 4.34 | ||
Jurkat | 55.4 | 0.54 | 5.54 | 5.42 |
Jurkat (old) | 104 | 0.29 | 10.4 | 2.88 |
- Mix 50/50 old/new Jurkat to make up 30ng
New: 2.71ul Old: 1.44ul
Sample | Volume | Make it up to 24ul |
NA12878 | 1.7 | 22.3 |
NA18507 | 2.42 | 21.58 |
NA20431 | 0.59 | 23.41 |
CV-Fibr | 6.2 | 17.8 |
CV-iPS-B | 4.13 | 19.87 |
CV-iPS-F | 4.34 | 19.66 |
Jurkat | 5.42 | 18.58 |
Jurkat (old) | 2.88 | 21.12 |
Jurkat (mix) | (NEW)2.71+(OLD)1.44 | 24 |
Prepare Master Mix
X1.05 | CHR22 | CHR21 | CHR8 |
2X iQ Super Mix | 189 | 189 | 189 |
Primer F (3.3uM) | 56.7 | 56.7 | 56.7 |
Primer R (3.3uM) | 56.7 | 56.7 | 56.7 |
- Aliquot 32ul to each well +8ul sample =40ul Total
Perform PCR reaction in real-time thermocycler
NA12878 | NA12878 | NA12878 | Jurkat (mix) |
NA18507 | NA18507 | NA18507 | Jurkat (mix) |
NA20431 | NA20431 | NA20431 | Jurkat (mix) |
CV-Fibr | CV-Fibr | CV-Fibr | |
CV-iPS-B | CV-iPS-B | CV-iPS-B | |
CV-iPS-F | CV-iPS-F | CV-iPS-F | |
Jurkat | Jurkat | Jurkat | |
Jurkat (old) | Jurkat (old) | Jurkat (old) | |
Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever