AlanFung:LabNotes/Capturing/BisFirstExon 2009 12 4: Difference between revisions

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>Alan6017518
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==qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)==
==qPCR Validation(3 Bisulfite Conversion Primers + gDNA 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 10ng||1/10 Dilution ng/ul||To get 10ng
| Sample||Sample Concentration ng/ul||To get 30ng||1/10 Dilution||To get 30ng
|-
|-
| NA12878||17.6||0.57||||
| NA12878||17.6||1.7||||
|-
|-
| NA18507||12.4||0.81||||
| NA18507||12.4||2.42||||
|-
|-
| NA20431||50.6||||5.06||1.98
| NA20431||50.6||0.59||||
|-
|-
| CV-Fibr||4.84||2.07||||
| CV-Fibr||4.84||6.2||||
|-
|-
| CV-iPS-B||7.27||1.38||||
| CV-iPS-B||7.27||4.13||||
|-
|-
| CV-iPS-F||6.91||1.45||||
| CV-iPS-F||6.91||4.34||||
|-
|-
| Jurkat||55.4||||5.54||1.81
| Jurkat||55.4||0.54||5.54||5.42
|-
|-
| Jurkat (old)||104||||10.4||0.96
| Jurkat (old)||104||0.29||10.4||2.88
|-
|-
|  
|  
|}
|}
*Mix 50/50 old/new Jurkat to make up 10ng
*Mix 50/50 old/new Jurkat to make up 30ng
  New: 0.905ul
  New: 2.71ul
  Old: 0.48ul
  Old: 1.44ul


{| {{table}}
{| {{table}}
| Sample||Volume||Make it up to 8ul
| Sample||Volume||Make it up to 24ul
|-
|-
| NA12878||0.57||7.43
| NA12878||1.7||22.3
|-
|-
| NA18507||0.81||7.19
| NA18507||2.42||21.58
|-
|-
| NA20431||1.98||6.02
| NA20431||0.59||23.41
|-
|-
| CV-Fibr||2.07||5.93
| CV-Fibr||6.2||17.8
|-
|-
| CV-iPS-B||1.38||6.62
| CV-iPS-B||4.13||19.87
|-
|-
| CV-iPS-F||1.45||6.55
| CV-iPS-F||4.34||19.66
|-
|-
| Jurkat||1.81||6.19
| Jurkat||5.42||18.58
|-
|-
| Jurkat (old)||0.96||7.04
| Jurkat (old)||2.88||21.12
|-
|-
| Jurkat (mix)||1.39||6.62
| Jurkat (mix)||(NEW)2.71+(OLD)1.44||24
|-
|-
|  
|  
|}
|}
==Prepare Master Mix==
==Prepare Master Mix==
{| {{table}}
{| {{table}}

Revision as of 19:21, 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

      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