We make test PCR to choose optimal quantity of cycles and amount of cDNA for amplification, and there are lines on 9 cycles and 12 cycles (same sample), but the line on 15 cycles disappear (also same sample). RNA is good (we validate it by TapeStation): no smear, no clipped RNA. Does anybody know why does it happen?
Anastasia Vasilyeva then there is significant volume variation between the 9, 12 and 15 cycles; this will have an impact on the heating/cooling rate of the samples during PCR (or put differently, you will have two variables: reaction volume and cycle number). Why don't you do an experiment with a single sample (say, 3 uL cDNA of the 7-well sample) and set up three different PCR reactions and instead of taking aliquots out, take one tube out at cycle 9, the other at 12 and the other at 15 and see what happens?
Dear Anastasia, in all #15 wells there is no band or there is a small smear. In my opinion, it could be a DNase contamination or there is a mistake in the preparation of these tubes. Maybe you can increase the enzyme amount and repeat experiment.
Best regards.
Repetition is needed. Most likely due to the handling error it seems.
:) no sample added ? repeat the gel if you have your pcr. or better repeat pcr make sure you add everything :)
Mert Sudagidan and Ramamoorthy Sankaranarayanan , I disagree; I find it hard to believe there is a handling error or a nuclease contamination that consistently affects the 15-cycle timepoints of nine different reactions while leaving all other timepoints untouched. Not only that, do you notice how in five of the six reactions the 9-cycle timepoint actually yields more product than the 12-cycle timepoint (hence suggesting that the disappearance of the amplicon by cycle 15 is not an error)?
Anastasia Vasilyeva , can you provide more details about your experiment? Does the target amplicon contains repetitive sequences? How did you take samples at different cycles, by withdrawing an aliquot from the same reaction, by setting up triplicate reactions and taking out the appropriate set when the target cycle was reached, or by repeating the same PCR three times but with different number of cycles? Is your PCR device a block-type machine?
At first I was inclined to think that this would be a case of the denatured template adopting some secondary structure leading to mispriming and shortening of the resulting amplicon as cycles accumulated and primers depleted, but the fact that the same thing happens in the 3 uL cDNA reactions (where yield is much lower) means this thing is independent of amplicon and primer concentration and thus made me think otherwise...
Alejandro Martin, we take cDNA and then take 3 aliquots of cDNA to the 3 tubes: 1, 3 and 10 µl. Then we make first PCR in 18 cycles (15 µl in each tube).
Next we take 2nd test PCR: take 1 µl of 1st PCR product, mix it with 19 µl of water (20 time dilution) and add 1 µl of diluted 1st PCR product to 24 µl of master mix. After 1st PCR step in 9 cycles we take 5 µl from tube for phoresis. Then we make additional 3 cycles (results in 12 cycles) with same tube and again take 5 µl for phoresis. And then again 3 additional cycles (results in 15 cycles) and 5 µl for phoresis. And make phoresis with all this aliquots.
What about repetitive sequences... It's TCR genes. Alpha and beta. And as I know they haven't such sequences) And yes. Our PCR machines are block-type machines)
Anastasia Vasilyeva then there is significant volume variation between the 9, 12 and 15 cycles; this will have an impact on the heating/cooling rate of the samples during PCR (or put differently, you will have two variables: reaction volume and cycle number). Why don't you do an experiment with a single sample (say, 3 uL cDNA of the 7-well sample) and set up three different PCR reactions and instead of taking aliquots out, take one tube out at cycle 9, the other at 12 and the other at 15 and see what happens?
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