I'm doing a point mutation using PCR, I have seen bands in DNA gel that are larger (10000 or 8000 bp) than my template plasmid (6000 bp). Does anyone know reason for this.
Hi Nishya,
Are you doing a direct site mutation or inserting a fragment? Did you cut your plasmid with restriction enzymes before the electrophoresis?
Zayda
Hi Zayda,
I'm doing a direct site mutation to the circular DNA.No any insertion, I haven't cut my plasmid either.
I'm using quickchange ll site directed mutagenesis kit
Thank you
Nishya,
You should cut the plasmid or linearized it in order to know the real size.
In your kit you should have a restriction enzyme specific for it.
Zayda
according to the kit protocol I used, I can directly use my circular DNA for PCR
Sorry Zayda, I missunderstood your question. I did cut the plasmid before electrophoresis.
Exactly Nishya, to do the PCR you dont have to cut.. but before the electrophoresis did you cut? You should cut to see the real size.
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