AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4: Difference between revisions
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Step6 72C, 5m | Step6 72C, 5m | ||
Step7 4C, Forever | Step7 4C, Forever | ||
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| Jurkat (old)||CV-iPS-F | |||
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| Jurkat (mix)|| | |||
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| Jurkat|| | |||
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| NA12878|| | |||
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| NA18507|| | |||
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| NA20431|| | |||
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| CV-Fibr|| | |||
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| CV-iPS-B|| | |||
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Revision as of 18:41, 4 December 2009
qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)
- Start with 10ng template
Sample | Sample Concentration ng/ul | To get 10ng | 1/10 Dilution ng/ul | To get 10ng |
NA12878 | 17.6 | 0.57 | ||
NA18507 | 12.4 | 0.81 | ||
NA20431 | 50.6 | 5.06 | 1.98 | |
CV-Fibr | 4.84 | 2.07 | ||
CV-iPS-B | 7.27 | 1.38 | ||
CV-iPS-F | 6.91 | 1.45 | ||
Jurkat | 55.4 | 5.54 | 1.81 | |
Jurkat (old) | 104 | 10.4 | 0.96 | |
- Mix 50/50 old/new Jurkat to make up 10ng
New: 0.905ul Old: 0.48ul
Sample | Volume | Make it up to 8ul |
NA12878 | 0.57 | 7.43 |
NA18507 | 0.81 | 7.19 |
NA20431 | 1.98 | 6.02 |
CV-Fibr | 2.07 | 5.93 |
CV-iPS-B | 1.38 | 6.62 |
CV-iPS-F | 1.45 | 6.55 |
Jurkat | 1.81 | 6.19 |
Jurkat (old) | 0.96 | 7.04 |
Jurkat (mix) | 1.39 | 6.62 |
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
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
1 | 2 |
Jurkat (old) | CV-iPS-F |
Jurkat (mix) | |
Jurkat | |
NA12878 | |
NA18507 | |
NA20431 | |
CV-Fibr | |
CV-iPS-B | |