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]
- Common linker sequence in every probe is: CTTCAGCTTCCCGATATCCGACGGTAGTGT (Porecca et al. 2007)
- 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