AlanFung:LabNotes/2014/2014-9-9
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Repeat qPCR w less cycle and PAGE gel with increased amplicon[edit]
- Pick 8 libraries to test the amplification condition, stop reaction at cycle 8
- Libraries to test 6T1, 6T2, 7T1, 7T2, PCT1, PCT2, NORM1-02 P1, NORM1-03 P1
- Take out 6T1, 6T2, 7T1, PCT1 at cycle 8 before reaching plateau based on previous PCR curve
- Take out 7T2, NORM1-03 at cycle 9 before reaching plateau based on previous PCR curve
- Take out PCT2, NORM1-02 at cycle 10 before reaching plateau based on previous PCR curve
Component | 1 Library | 8 Libraries + 3% |
qPCR Master Mix: | ||
Adapter ligated DNA | 1 | 8.2 |
10uM PCR_F | 0.5 | 4.1 |
10uM PCR_R.Ind2 | 0.5 | 4.1 |
2X Kapa Fast | 12.5 | 103.0 |
Water | 10.5 | 86.5 |
Total master mix volume | 25 | 206.0 |
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold File:09092014 Cycle8.JPG File:09092014 Cycle9.JPG File:09092014 Cycle10.JPG
- PAGE TBE Gel verification w 5ul amplicon
File:ZhangLab 2 2014-09-09 15hr 13min.jpg
- result looks better than the over saturated adapter-primer dimer, library bands still look dim
- No idea why the beads purification after adapter ligation didn't remove the adapter-primer dimer, I ran a 1X bead purification for that step
To Do[edit]
- Perform another round of 1x bead purification to remove adapter-primer dimer
- Bisulfite Conversion
- 2 Rounds of PCR for cfDNA due to low 10ng input
- 1 round of PCR for tumor samples for we started off with 100ng input