Noi/NOTES/2011-9-4

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Revision as of 17:31, 5 September 2011 by >Noi (→‎PCR set up)
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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
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.