Biology-Online • View topic - Load DNA PCR Product into Agarose Gel
Login

Join for Free!
121818 members
Advertisement
Advertisement

Load DNA PCR Product into Agarose Gel

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

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.
9afa
Garter
Garter
 
Posts: 6
Joined: Sat Nov 10, 2012 4:05 pm

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.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

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 ???
9afa
Garter
Garter
 
Posts: 6
Joined: Sat Nov 10, 2012 4:05 pm


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.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Postby 9afa » Sun Nov 11, 2012 10:44 am

Ahaa, so do you think that will affect the sequencing??
9afa
Garter
Garter
 
Posts: 6
Joined: Sat Nov 10, 2012 4:05 pm

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/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

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 .
9afa
Garter
Garter
 
Posts: 6
Joined: Sat Nov 10, 2012 4:05 pm

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.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Postby 9afa » Tue Nov 13, 2012 1:05 pm

amplicon
9afa
Garter
Garter
 
Posts: 6
Joined: Sat Nov 10, 2012 4:05 pm

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/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

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

Ok thanks :)
9afa
Garter
Garter
 
Posts: 6
Joined: Sat Nov 10, 2012 4:05 pm


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 0 guests