Revision as of 01:00, 8 January 2010 by >Alan6017518
Qubit Quantification (ssDNA)[edit]
- Prepare 4 Qubit Assay Tubes (2 for ladder, 2 for samples)
- Need 800ul Diluted Dye
Mix 4ul dye in 796 Quant -iT buffer
- Mix 10ul standard with 190 ul working soln.
- Mix 2ul sample with 198ul working soln.
- Vortex all tubes for 2-3 secs
- Incubate for 2 mins at RT
CT Converted Jurkat 1.1ng/ul * 20ul = 22ng
CT Converted CV-Fibr 16.2ng/ul * 20ul = 324ng
|
Start (ng) |
End (ng) |
Yield %
|
CV-Fibr |
500 |
324 |
64.8
|
Jurkat |
100 |
22 |
22
|
|
Yield is lower than expected, Jurkat DNA might be degraded already.
qPCR Validation(3 Bisulfite Conversion Primers)[edit]
- Start with 10ng template for each set of primer (30ng Total)
|
Concentration |
Volume |
Make up to 24ul
|
CV-Fibr |
16.2 |
1.85 |
22.15
|
Jurkat |
1.1 |
10 |
14
|
|
Prepare SuperMix[edit]
- Prepare 3 tubes for 3 different primers mix
|
CHR22 |
CHR21 |
Chr8
|
2X iQ Super Mix |
44 |
44 |
44
|
Primer F (3.3uM) |
13.2 |
13.2 |
13.2
|
Primer R (3.3uM) |
13.2 |
13.2 |
13.2
|
|
- Aliquot 32ul to each well +8ul sample =40ul Total
Perform PCR reaction in real-time thermocycler
Setup
|
CV-Fibr (CHR22)
|
CV-Fibr (CHR21)
|
CV-Fibr (CHR8)
|
Jurkat (CHR22)
|
Jurkat (CHR21)
|
Jurkat (CHR8)
|
|
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
8ul h2o+1.5ul 6x loading dye+0.5ul 25bp ladder
6.5ul h2o+1.5ul 6X loading dye+2ul sample
Run at 250V for 20min
Lane |
Setup |
|
1 |
LM Ladder |
|
2 |
CV-Fibr (CHR22) |
|
3 |
CV-Fibr (CHR21) |
|
4 |
CV-Fibr (CHR8) |
|
5 |
Jurkat (CHR22) |
|
6 |
Jurkat (CHR21) |
|
7 |
Jurkat (CHR8) |
|
8 |
NA12878 |
PCR_AMP F/R 6.3r Sol
|
9 |
NA18507 |
PCR_AMP F/R 6.3r Sol
|
10 |
NA20431 |
PCR_AMP F/R 6.3r Sol
|
11 |
Jurkat_New |
PCR_AMP F/R 6.3r Sol
|
12 |
Jurkat _Old |
PCR_AMP F/R 6.3r Sol
|
|
File:ZhangLab 2 2010-01-07 14hr 55min.jpg
- CT Conversion Works
- PCR with sequencing adapters works on NA20431, mix NA20431 (6.3 & 6.3r Sol) and perform size selection
Qiaquick[edit]
- Mix the PCR amplified products of NA20431 together
- Purify with 2X Qiaquick columns, eluted each with 22 ul EB.
- Measure concentration with nanodrop
Result: 64.4ng/ul * 44ul = 2833.6ng
Size Selection[edit]
- For optimal results load up to 700ng of total sample per well (2833.6/700=Run 4 wells)
- Fill any unused well with 30ul EB Buffer
- Mix 15ul EB buffer with 0.5ul LM bp ladder in a 0.2ml tube
- Load 20ul end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
- Run the SizeSelect program for 12~14 min. Pause when the band just move into the middle collection well
- Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
- Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
- Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.
File:ZhangLab 2 2010-01-07 16hr 53min.jpg