Load DNA PCR Product into Agarose Gel

[9afa]

Dear Scientist

I am a new member and I have A question which I would like to share it with you to help me to get the reason.

I am asking why do I got double band align under each other in Agarose Gel when I load DNA PCR Product ???
Knowing that this DNA is Human DNA and I got it from Buccel swab and I am working in SNPs sequencing.

Thanks in Advance.

[JackBean]

What was your PCR DNA template? Did you check specificity of your PCR? Are you sure there is no splicing?

[9afa]

Thanks a lot for your quick reply JackBean.

Yes, I check all of what you said and It works at the first time without any problems, just suddenly it appeared without changing any things except I increased No. of cycling to 35 instead of 30 so is that a big deal and may appear as a double band in Agarose Gel ???

[JackBean]

well, in such case, it could be that you had that band also before, it just wasn't visible. The amplification was not that strong, but after 5 more circles the amount of DNA is 32-times higher.

[9afa]

Ahaa, so do you think that will affect the sequencing??

[JackBean]

depends what you want to sequence. Will you cut your amplicon and purify before sequencing?

[9afa]

mmmm, I think yes because I did a PCR for particular reagion by adding a specific primer (exon) before sequencing .

[JackBean]

but what is your teplate for sequencing? Is it plasmid DNA or the amplicon from PCR which have you purified from gel?

[9afa]

amplicon

[JackBean]

so you run it on gel, cut it from it and clean? Than it should be fine.

[9afa]

Ok thanks