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Load DNA PCR Product into Agarose Gel

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Load DNA PCR Product into Agarose Gel

Postby 9afa » Sat Nov 10, 2012 4:21 pm

Dear Scientist :)

I am a new member :D 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.
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Postby JackBean » Sat Nov 10, 2012 4:25 pm

What was your PCR DNA template? Did you check specificity of your PCR? Are you sure there is no splicing?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby 9afa » Sat Nov 10, 2012 6:09 pm

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 ???
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Postby JackBean » Sat Nov 10, 2012 6:37 pm

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.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby 9afa » Sun Nov 11, 2012 10:44 am

Ahaa, so do you think that will affect the sequencing??
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Postby JackBean » Sun Nov 11, 2012 1:11 pm

depends what you want to sequence. Will you cut your amplicon and purify before sequencing?
http://www.biolib.cz/en/main/

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Postby 9afa » Mon Nov 12, 2012 10:58 am

mmmm, I think yes because I did a PCR for particular reagion by adding a specific primer (exon) before sequencing .
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Postby JackBean » Mon Nov 12, 2012 2:53 pm

but what is your teplate for sequencing? Is it plasmid DNA or the amplicon from PCR which have you purified from gel?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby 9afa » Tue Nov 13, 2012 1:05 pm

amplicon
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Postby JackBean » Tue Nov 13, 2012 3:24 pm

so you run it on gel, cut it from it and clean? Than it should be fine.
http://www.biolib.cz/en/main/

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Postby 9afa » Sat Nov 17, 2012 2:06 pm

Ok thanks :)
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