Revision as of 18:39, 4 December 2009 by >Alan6017518
qPCR Validation(3 Bisulfite Conversion Primers + gDNA primers)
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
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