Noi/NOTES/2011-9-4
Jump to navigation
Jump to search
Randomly tagging primer test
- continued from 2011_09_02: [[1]]
Tests:
- Do the second set primers, Syb_FP5A & Syb_RP7, can amplify the amplicon amplified by the first primer set, AmpF7AUSol and AmpR6.3Sol?
- To test this I used the DNA template that expected to be amplified by these primers
- DMR330k SeqLib (GA samples), used 2ng in 50 ul reaction
- + control amplicon amplified by the first primer set conc. 0.1uM, used 2ul of 100x diluted PCR product in total 50ul
- I also tested to make sure that these primers can not amplify the captured DNA by, used captured DNA as the DNA template
PCR set up
DNA templates |
1. NTC |
2. DMR330k-SeqLib (GA sample) |
3. + control-0.1uM-no USER |
4. Circular DNA |
Components | 1 rxn | 4.2x rxn mix |
DNA template | 2.00 | 0.00 |
10 uM Syb_FP5A | 1.00 | 4.20 |
10uM Syb_RP7 | 1.00 | 4.20 |
2x KAPA | 25.00 | 105.00 |
H2O | 21.00 | 88.20 |
Total volume (ul) | 50.00 | 210.00 |
- aliquot 48ul, add 2ul DNA template
Program
98C 30s -> (98C 10s -> 55C 20s -> 72C 20s)x25-> 72C 3min->15C hold
File:QPCR-test-Syb-primers-20cycles-edit.png
- From the realtime-PCR curves, the second set primers can amplify either the sequencing library prepared by library-free primer and the amplicon amplified by new primers. Next I can test the amplification by these primers of the reaction amplified by randomly tagging primers for 2 cycles and after USER digestion.
- In addition, there was no amplification in the reaction contained only captured DNA.