AlanFung:LabNotes/Capturing/BisFirstExon 2010 1 7
Jump to navigation
Jump to search
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
Result[edit]
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.