Revision as of 00:23, 5 December 2009 by >Alan6017518
qPCR Validation(3 Bisulfite Conversion 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 |
19.85
|
|
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
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
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)