Hi everyone,
I have been struggling with a PCR amplification for weeks now. First, let me explain to you the context:
I'm amplyfing a gene for a subsequent cloning (the gene is 2.5kb, arabidopsis)
I'm using a Phusion ploymerase.
Now here comes the incredible thing.
The first 4 PCR amplified my gene perfectly, but I needed to do more. From then, I always get a terrible smear (the one in the picture)
I tried EVERYTHING.
New primers, new reagents, new cDNA, different concentration, 2 different buffers (HF and GC), different temperatures, different PCR machines.
NOTHING seems to work, but why it did in the first place?!
Has this ever happened to some of you? What could I do?
Thanks for the support
Which one of these samples is the no template control?
This looks like you have post pcr contamination in your assay and everything is reamplifying the contamination so you have huge over amplification. Try running a pcr with no added dna and see if you get the same result
Hii,
What concentrations of dNTPs, primers and template DNA you are using. Also, as emphasized by Dr. Paul Rutland, try to maintain a negative control while performing this experiment next time.
You may try Hot-start PCR, and use diluted cDNA (1/10 dil. for e.g.) for amplification.
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