Noi/NOTES/2011-9-4

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Randomly tagging primer test[edit]

  • continued from 2011_09_02: [[1]]

Part I[edit]

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 for 50cycles, 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 using captured DNA as the DNA template

PCR set up[edit]

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 for 50 cycles. This means that the second set primers works well. Next, I will 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 that contained only captured DNA. It confirmed that the primers amplified 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.

Part II[edit]

  • 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 (2011_09_02), 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 primers 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 also vary the volume of USER (1, 2 & 5ul) even in the previous experiment I can not see the amplification after USER digestion. This just wants to confirm again that USER can completely digest the uracil containing primers and 1ul of USER is good enough.

PCR set up For the first 2 cycles[edit]

  • Note: All reactions used captured DNA as the template

A: 0.2uM primers[edit]

PCR conditions code
1. NTC-0.2uM-1ul USER N-10
2. + control-0.2uM-no USER .+10
3. Exp-0.2uM-1ul USER E10-1
4. Exp-0.2uM-2ul USER E10-2
5. Exp-0.2uM-5ul USER E10-5
Components 1 rxn 5.2 rxn mix A
Circular DNA 2.00 0.00
10uM AmpF7AUSol 1.00 5.20
10uM AmpR6.3Sol 1.00 5.20
2x KAPA 25.00 130.00
H2O 21.00 109.20
Total volume (ul) 50.00 260.00
  • aliquot 48ul, add 2ul circular DNA

B: 0.1uM primers[edit]

PCR conditions code
1. NTC-0.1uM-1ul USER N-5
2. + control-0.1uM-no USER .+5
3. Exp-0.1uM-1ul USER E5-1
4. Exp-0.1uM-2ul USER E5-2
5. Exp-0.1uM-5ul USER E5-5
Components 1 rxn 5.2 rxn mix B
Circular DNA 2.00 0.00
10uM AmpF7AUSol 0.50 2.60
10uM AmpR6.3Sol 0.50 2.60
2x KAPA 25.00 130.00
H2O 22.00 114.40
Total volume (ul) 50.00 260.00
  • aliquot 48ul, add 2ul circular DNA

C: 0.04uM primers[edit]

PCR conditions code
1. NTC-0.04uM-1ul USER N-1
2. + control-0.04uM-no USER .+1
3. Exp-0.04uM-1ul USER E1-1
4. Exp-0.04uM-2ul USER E1-2
5. Exp-0.04uM-5ul USER E1-5
Components 1 rxn 5.2 rxn mix B
Circular DNA 2.00 0.00
1uM AmpF7AUSol 2.00 10.40
1uM AmpR6.3Sol 2.00 10.40
2x KAPA 25.00 130.00
H2O 19.00 98.80
Total volume (ul) 50.00 260.00
  • aliquot 48ul, add 2ul circular DNA

D: 0.02uM primer[edit]

PCR conditions code
1. NTC-0.02uM-1ul USER N-1
2. + control-0.02uM-no USER .+1
3. Exp-0.02uM-1ul USER E1-1
4. Exp-0.02uM-2ul USER E1-2
5. Exp-0.02uM-5ul USER E1-5
Components 1 rxn 5.2 rxn mix B
Circular DNA 2.00 0.00
1uM AmpF7AUSol 1.00 5.20
1uM AmpR6.3Sol 1.00 5.20
2x KAPA 25.00 130.00
H2O 21.00 109.20
Total volume (ul) 50.00 260.00
  • aliquot 48ul, add 2ul circular DNA

Program

  • 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x2 -> 72C 5min -> 4C
  • Add USER -> 37C 2hr -> 70C 20min
  • Prepare 1xKAPA with 0.4uM Syb F/R primers, after adding equal volume of reaction mix to the previous reaction (50ul+50ul), the final conc. of the second set primers is 0.2 uM
Components 1 rxn 21.00
10uM Syb_FP5A 2.00 42.00
10uM Syb_RP7 2.00 42.00
2x KAPA 25.00 525.00
H2O 21.00 441.00
Total 50.00 1050.00
  • Add 50 ul of 1x KAPA with primer mix to each reaction

Program 98C 30s -> (98C 10s -> 55C 20s -> 72C 20s)x33 cycles ->72C 3min ->15C hold (actually set up at 50 cycles)
File:0.2uMprimer-qPCR-curve.pngFile:0.1uMprimer-qPCR-curve.pngFile:0.04uMprimer-rPCR-curve.pngFile:0.02uMprimer-qPCr-curve.png

  • From the results, the second set primers can successfully amplify the DNA amplified by randomly tagging primers and digested with USER. In every condition, we saw the +control showed better amplification than the reaction with USER digestion. This because the uracil containing primers and DNA were not digested. These results were consistent in every primer concentration.
  • There was no different between the reaction digested with 1, 2 or 5 ul of USER, so 1 ul is good in the next experiment.
  • There was amplification in the reaction that started with 0.02uM primers in the first 2 cycles. However the Ct value was pretty high ~28.5. Note: the tube with 5ul USER showed realtime-PCR strange from 1 and 2ul. I guess it might be some mistake during adding reagents because other condition showed consistent result.

Next:

  • Decide what's randomly tagging primers concentration in the first two cycles?
  • Perform clonal sequencing to verify if we get tagging primer left after USER digestion -> analyze Sanger sequencing -> try with real samples that can be used for the manuscript.

  • continued on 2011_09_05: [[2]]