Matt:LabNotes/2013-7-1

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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-28

Amplification with Sequencing Adapters[edit]

  • Since this is the same design as LC Sciences probes that Noi used, I'll use the same primers
Tube # Sample Index#
1 NTC-0gap Indx7
2 gDNA-0gap Indx45
3 cDNA-0gap Indx76
4 NTC-20gap Indx7
5 gDNA-20gap Indx77
6 cDNA-20gap Indx78


Will also do duplicate for each sample so 12 reactions total

Components 1 rxn 13 rxn
Captured template 12.00 0.00
10uM Forward+Indx 2.00 0.00
10uM Reverse 2.00 26.00
2x KAPA SYBG fast MM 50.00 650.00
H2O 34.00 442.00
Total 100.00 1300.00
 Program
 *98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x7 cycles -> 72°C 3 min -> 15°C hold

File:07012013 Agi26kCapturedSequencePCRGraph.JPG

  • Sample with greatest amplification is NTC for 20gap probes
    • Will run a gel to see what is causing the signal
  • Both 20gap samples did not show amplification suggesting something went wrong
    • Possibly the dNTP mix used during capture reaction is to blame because Noi says the Stoffel fragment enzyme was working a week ago
    • Will do a PCR with the same dNTP mix to see
  • Both 0gap samples showed moderate amplification and NTC was as expected

PCR'd an additional 3 cycles to make a total of 21 to match CustomArray

 Program
 *98°C 30sec -> (98°C 10sec -> 72°C 2min30sec) x3 cycles

Gel Check[edit]

File:2013-07-01 Agi26kCapturedPCR GelCheck.jpg

  • Looks like I mixed up 20gap samples while pipetting but I'm not sure how
    • I was very careful about adding correct captured template to each PCR reaction
    • If this was caused by switching two samples, then expect to see two bright bands and one dim/nonexistent one
    • Also each sample is consistent with its duplicate in qPCR curve, and it is unlikely I would pipette incorrectly twice
    • Also a faint band around 300bp can still be seen in samples (possibly will be brighter after 3 more amplification cycles)
  • It is possible a mistake happened during capture reaction but I was also very careful with putting reagents in the right tubes during that step
  • Another possibility is that NTC-20gap had contamination somehow and everything else works fine
    • Will know more after testing dNTP mix tomorrow

Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-2