Question: (Closed) PCR with 2 cycles gave no products
gravatar for ramonaramonawan
4.0 years ago by
ramonaramonawan10 wrote:

Hi guys, As I am going to do the Miseq sequencing. I wanted to run the 1st PCR with 2 cycles, and input the 1st PCR product to the 2nd PCR, which I can run over 20 cycles (I need to test the minimum cycle number of course).

To confirm this strategy works, I used 50ng genomic DNA, run the PCR with 40nM primer each, for 2 cycles (total 25ul volume). I also ran 32 cycles as a positive ct to ensure the PCR works. Later, I input 1ul, 5ul, 12.5ul and 25ul of the PCR product from the 1st PCR to the second PCR, with 500nM primers and 35 cycles. However, I could not get any band from this reaction, except that I took 1ul from the 1st PCR with 32 cycles, and input for the 2nd PCR which gave a nice band.

So any suggestions?

pcr • 975 views
ADD COMMENTlink written 4.0 years ago by ramonaramonawan10

Hello ramonaramonawan!

We believe that this post does not fit the main topic of this site.

Not a bioinformatics question. Alternative sites

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.


ADD REPLYlink modified 4.0 years ago • written 4.0 years ago by Michael Dondrup48k
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