Matt:LabNotes/2013-6-6

From ZhangLabWiki
Jump to navigation Jump to search

Sequence Agilent Oligos[edit]

  • Diluted Expansion PCR'd 0 and 20 gap oligos from 112nM and 123 nM, respectively, to 50ul of 10nM each in a separate tube

PCR[edit]

PCR Mix

Reaction of 0 gap probes:

Components Volume
10nM Agilent Expansion PCRd 0 gap Oligos 0.40
100uM Forward ISB_AP1V6_AFv1 0.40
100uM Reverse ISB_AP2V6_AR 0.40
2x KAPA SYBG fast MM 50
H2O 49
Total 100.2

Reaction of 20 gap probes:

Components Volume
10nM Agilent Expansion PCRd 20 gap Oligos 0.40
100uM Forward ISB_AP1V4_AFv1 0.40
100uM Reverse ISB_AP2V4_AR 0.40
2x KAPA SYBG fast MM 50
H2O 49
Total 100.2

Agilent Oligos: Expansion PCRd but before Production PCR (see: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-10)

Program 98C 30s -> (98C 5s -> 50C 20s -> 72C 30s)x5 -> (98C 5s -> 72C 30s)x10 -> 72C 3min

  • Did same number of PCR cycles as Production PCR to keep amplification as consistent as possible

Dan's Probes[edit]

  • Set up reaction for his probes the same as for 0 gap and 20 gap Agilent probes with same volumes of probe, primer, H2O and KAPA MM
  • Took out Dan's samples after 13 cycles
  • Because Dan's had more oligos than I expected (4 sets, each set with V4 and V6 within them), I had to repeat sequencing barcodes and so assigned them as follows:
Tube # Probes (0.4ul) Forward Primer 100uM (0.4ul) Reverse Primer 100uM (0.4ul) Total Cycles
1 0gap 10nM ISB_AP1V6_AFv1 ISB_AP2V6_AR 15
2 20gap 10nM ISB_AP1V4_AFv1 ISB_AP2V4_AR 15
3 #1-V6 20nM ISB_AP1V6_AFv2 ISB_AP2V6_AR 13
4 #1-V4 20nM ISB_AP1V4_AFv2 ISB_AP2V4_AR 13
5 #2-V6 20nM ISB_AP1V6_AFv1 ISB_AP2V6_AR 13
6 #2-V4 20nM ISB_AP1V4_AFv1 ISB_AP2V4_AR 13
7 #3-V6 20nM ISB_AP1V6_AFv2 ISB_AP2V6_AR 13
8 #3-V4 20nM ISB_AP1V4_AFv2 ISB_AP2V4_AR 13
9 #4-V6 20nM ISB_AP1V6_AFv1 ISB_AP2V6_AR 13
10 #4-V4 20nM ISB_AP1V4_AFv1 ISB_AP2V4_AR 13
 *First made 9X Master Mix of KAPA and H2O: 450ul KAPA + 441ul H2O

File:060613 AgiOligoSeq qPCR.JPG File:060613 DansOligoSeq qPCR.JPG

20 gap Probe Capture Reaction[edit]

Followed plan from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4