Alice:LabNotes/2009-12-3
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Sanger validation of candidate mutations[edit]
DNA preparation[edit]
- prepared the following stock solutions for PCR templates
Sample Conc. Volume H2O Final Conc. CV-iPS F 309ng/ul 1ul 153.5ul 2ng/ul CV-ips B 358ng/ul 1ul 178ul 2ng/ul CV-Fibr 712ng/ul 1ul 355ul 2ng/ul
PCR[edit]
- Primers and templates:
' | CV-fibroblast | CV-ips-B | CV-ips-F |
MCM3 | x | x | x |
18-22778270 | x | ||
ZSCAN | x | x | x |
C1orf | x | x | x |
SPEN | x | x | x |
- 13 reactions total.
DNA(2ng/ul) 3ul Taq 2x master mix 25ul 10uM Primer mix 1ul H2O 21ul total 50ul 94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min.
- use 3ul to run a TBE gel to check the PCR results, then save 22ul for backup, and use the rest to do Qiaquick column purification elute in 30ul EB.
- Use Nanodrop to check the concentration of the purified PCR product.
- TBE gel result:
File:ZhangLab 2 2009-12-04 10hr 21min.jpg File:ZhangLab 2 2009-12-03 15hr 14min.jpg
- Prepare the samples for sequencing:
- USE Pre-Mixed Samples Preparation Guidelines from Genewiz to mix the DNA and custom primers.
- The DNA size is 200-500bp, so we use 3-10ng of DNA in 8ul volume
' | CV-fibroblast (DNA+H2O) | CV-ips-B (DNA+H2O) | CV-ips-F (DNA+H2O) |
MCM3 | 1ul + 7ul | 2ul + 6ul | 1ul + 7ul |
18-22778270 | 1ul + 7ul | ||
ZSCAN | 1.2ul + 6.8ul | 1.5ul + 6.5ul | 1.5ul + 6.5ul |
C1orf | 1ul + 7ul | 1.1ul + 6.9ul | 1.2ul + 6.8ul |
SPEN | 2ul + 6ul | 1.1ul + 6.9ul | 2ul + 6ul |
- Add the Forward primer for sequencing, dilute the Forward primer to 2uM, add 4ul of primer and get a final volume of 12ul.
- [[Media:|Raw ABI trace file]].