Noi/NOTES/2011-9-4
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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 (100x diluted) |
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 both the sequencing library prepared by library-free primers and the amplicon amplified by randomly tagging primers. Next, I can test these primers with the DNA template 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. It confirmed that the primer will amplify specifically to the DNA amplified by the first primer set not the circulalr DNA.
- Note:
- Since this experiment was performed to test whether the primers work or not, the amount of DNA template in each reaction was not adjusted in the same amount.
- In the next experiment, I plan to test the amplification by the second set primers with the DNA amplified by different concentration of randomly tagging primers and also vary the amount of USER.
From the previous experiment, even we can not see the amplification by the lowest concentration primers (0.02uM), but I still test this condition, The reason was that in the previous experiment the concentration of primers was kept very low at 0.02uM for the whole procedure. But in the actual experiment, the second set primer will be added after the 2nd cycle of amplification and the concentration is much higher at 0.2uM. We might see better amplification in this condition when compare to the previous experiment.
- I vary