AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4: Difference between revisions
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>Alan6017518 (New page: ==qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)== *Start with 10ng template {| {{table}} | Sample||Sample Concentration ng/ul||To get 10ng||1/10 Dilution ng/ul||To get 10n...) |
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==qPCR Validation(3 Bisulfite Conversion Primers | ==qPCR Validation(3 Bisulfite Conversion Primers)== | ||
*Start with 10ng template | *Start with 10ng template for each set of primer (30ng Total) | ||
{| {{table}} | {| {{table}} | ||
| Sample||Sample Concentration ng/ul||To get | | Sample||Sample Concentration ng/ul||To get 30ng||1/10 Dilution||To get 30ng | ||
|- | |- | ||
| NA12878||17.6|| | | NA12878||17.6||1.7|||| | ||
|- | |- | ||
| NA18507||12.4|| | | NA18507||12.4||2.42|||| | ||
|- | |- | ||
| NA20431||50.6|||| | | NA20431||50.6||0.59|||| | ||
|- | |- | ||
| CV-Fibr||4.84||2 | | CV-Fibr||4.84||6.2|||| | ||
|- | |- | ||
| CV-iPS-B||7.27|| | | CV-iPS-B||7.27||4.13|||| | ||
|- | |- | ||
| CV-iPS-F||6.91|| | | CV-iPS-F||6.91||4.34|||| | ||
|- | |- | ||
| Jurkat||55.4||||5.54||1. | | 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 | |||
{| {{table}} | |||
| 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||19.85 | |||
|- | |||
| | |||
|} | |||
==Prepare Master Mix== | |||
{| {{table}} | |||
| 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 | |||
{| {{table}} | |||
| CHR22||CHR21||CHR8|| | |||
|- | |||
| NA12878||NA12878||NA12878||Jurkat (mix) CHR22 | |||
|- | |||
| NA18507||NA18507||NA18507||Jurkat (mix) CHR21 | |||
|- | |||
| NA20431||NA20431||NA20431||Jurkat (mix) CHR8 | |||
|- | |||
| 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 | |||
==TBE Gel Verification== | |||
*Run 3 10 well gels | |||
Gel 1 | |||
Ladder NA12878(22) NA12878(21) NA12878(8) NA18507(22) NA18507(21) NA18507(8) NA20431(22) NA20431(21) NA20431(8) | |||
Gel 2 | |||
Ladder CV-Fibr(22) CV-Fibr(21) CV-Fibr(8) CV-iPSB(22) CV-iPSB(21) CV-iPSB(8) CV-iPSF(22) CV-iPSF(21) CV-iPSF(8) | |||
Gel 3 | |||
Ladder Jurkat(22) Jurkat(21) Jurkat(8) Jurkat_Old(22) Jurkat_Old(21) Jurkat_Old(8) Jurkat_Mix(22) Jurkat_Mix(21) Jurkat_Mix(8) | |||
15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder | |||
15ul h2o+3ul 6X loading dye+2ul sample | |||
Run at 250V for 20min | |||
[[Image:ZhangLab_2 2009-12-04 17hr 36min.jpg|300px]] | |||
[[Image:ZhangLab_2 2009-12-04 18hr 08min.jpg|300px]] | |||
[[Image:ZhangLab_2 2009-12-04 18hr 05min.jpg|300px]] |
Latest revision as of 02:23, 5 December 2009
qPCR Validation(3 Bisulfite Conversion Primers)[edit]
- 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 | 19.85 |
Prepare Master Mix[edit]
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
CHR22 | CHR21 | CHR8 | |
NA12878 | NA12878 | NA12878 | Jurkat (mix) CHR22 |
NA18507 | NA18507 | NA18507 | Jurkat (mix) CHR21 |
NA20431 | NA20431 | NA20431 | Jurkat (mix) CHR8 |
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
TBE Gel Verification[edit]
- Run 3 10 well gels
Gel 1 Ladder NA12878(22) NA12878(21) NA12878(8) NA18507(22) NA18507(21) NA18507(8) NA20431(22) NA20431(21) NA20431(8)
Gel 2 Ladder CV-Fibr(22) CV-Fibr(21) CV-Fibr(8) CV-iPSB(22) CV-iPSB(21) CV-iPSB(8) CV-iPSF(22) CV-iPSF(21) CV-iPSF(8)
Gel 3 Ladder Jurkat(22) Jurkat(21) Jurkat(8) Jurkat_Old(22) Jurkat_Old(21) Jurkat_Old(8) Jurkat_Mix(22) Jurkat_Mix(21) Jurkat_Mix(8)
15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder 15ul h2o+3ul 6X loading dye+2ul sample Run at 250V for 20min
File:ZhangLab 2 2009-12-04 17hr 36min.jpg File:ZhangLab 2 2009-12-04 18hr 08min.jpg File:ZhangLab 2 2009-12-04 18hr 05min.jpg