Login

Join for Free!
119233 members


Purifying Recombinant plasmid DNA

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

Moderator: BioTeam

Purifying Recombinant plasmid DNA

Postby xbritoe » Mon Jan 16, 2012 8:23 am

Hi, so I have been transforming recombinant plasmid DNA into E.coli bacteria to clone the DNA. The plasmid consist of an ampicillin resistant gene. After I grow the bacteria, I would purify the plasmid DNA out of the bacterial cell using a maxi prep kit. So I was wondering if the final product of my purification consist of plasmid DNA (including the AMP resistant gene) or does it only consist of the DNA fragment that I initially placed into the plasmid.


I have been using my cloned DNA to transfect a cell line, so I was just wondering if I have been transfecting it with only my target gene or if I have been transfecting the whole plasmid including the AMP-R gene.

Thank you
xbritoe
Garter
Garter
 
Posts: 2
Joined: Mon Jan 16, 2012 8:05 am

Postby JackBean » Mon Jan 16, 2012 9:10 am

the kit extracts any intact DNA molecule of particular size, thus you will obtain whole plasmid
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5692
Joined: Mon Sep 14, 2009 7:12 pm

Re: Purifying Recombinant plasmid DNA

Postby JackBean » Mon Jan 16, 2012 9:12 am

xbritoe wrote:I have been using my cloned DNA to transfect a cell line, so I was just wondering if I have been transfecting it with only my target gene or if I have been transfecting the whole plasmid including the AMP-R gene.

Shouldn't you be the one who knows? Depends on what cell line and what was the protocol, you could either use whole plasmid or only part of it. But the transformation was probably based on recombination, thus you should have been transfecting only your gene plus something extra.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5692
Joined: Mon Sep 14, 2009 7:12 pm



Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 1 guest