AlanFung:LabNotes/2014/2014-9-9

From ZhangLabWiki
Jump to navigation Jump to search

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