After performing symmetric PCR, PCR purification was performed.
Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the agarose gel, and only a single band of dsDNA was confirmed.
Referring to other papers, I adjusted the annealing temperature, buffer composition, amount of primer, and amount of template, but the result was the same.
The primers were maintained at a ratio of 50:1 with 200 pmol of F primer and 4 pmol of R primer in 100uL of PCR contents, and TBE gel electrophoresis was also attempted, but the result was the same as the agarose gel. PCR performed without adding R primer showed a PCR band that was completely different in size from the template, and this seemed to be non-specific amplification.
I have never confirmed ssDNA and dsDNA at the same time in an asymmetric PCR result.
'Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners'
Referring to the following paper, we synthesized the same sequence and performed under the same conditions. The only difference was that the company of taq we used was different.
Since the PCR equipment was also new, it should not be a problem with the equipment.
Please let me know if you can help me. Thank you.
If you get a product with only one primer but of apparently the wrong size this may still be the right product. ssDNA forms different conformations compared with dsDNA so may run at an apparently larger size than a more tightly coled dsDNA so your product may not be non-specific. Try more single primer cycles to see if the band increases in intensity and use a small amount of the ss dna in a 2 primer normal pcr to see if it generates the right size dsDNA band again
Hello, Professor.
First of all, thank you for your answer.
The results of Asymmetric PCR with a small amount of R primer and a large amount of F primer and Asymmetric PCR with only F primer have already been confirmed by TBE-PAGE.
If you are familiar with Asymmetric PCR, can you tell me the protocol for how you performed the experiment to confirm the ssDNA band?
If you tell me, I would like to follow it.
Also, when performing Asymmetric PCR, the contents are adjusted to 100 uL in total, and I am curious about how many ng of the purification product (template) is used when performing Asymmetric PCR by purifying the symmetric PCR product.
I am also curious about the amount of F and R primers.
I referred to the paper mentioned in the text and performed PCR with 1.6 ng of template in 100 uL of Asymmetric PCR, and only dsDNA was observed at that time, so I thought I misunderstood the paper.
So I performed Asymmetric PCR with 160 ng of template, but only dsDNA bands were observed at 30 cycles, which was similar for both results.
I never used the 2 primer method. It was enough to just add one primer to the dsDNA amplimer and run a lot of cycles because each cycle only prduces the same amount as the starting material so for single primer amplification 40-50 cycles used to work fine but a better method is to phosphorylate the 5' end of the primer on the strand that you do not want ,then pcr then digest the pcr product with lambda exonuclease which will leave the strand that you do want. Phosphorylation is best done during the synthesis of the oligo commercially
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