shRNA ligation

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

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
Dagon123
Garter
Garter
Posts: 2
Joined: Wed Mar 23, 2011 4:55 pm

shRNA ligation

Post by Dagon123 » Wed Mar 23, 2011 5:07 pm

Hello, i'm new here.
I've been trying to subcloning a shRNA (60bp) into a empty vector. I don't have any problem with the transformation. When i try to check the positive colonies, i digest the vector with two restriction enzymes to find a fragment of about 360bp and the empty vector should give about 300bp.
my question is if 60bp are enough to discriminate two bands on a agarose gel to confirm the insertion of the fragment? or maybe.., there is another way to do this

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Wed Mar 23, 2011 5:24 pm

On the correct gel (high agarose, maybe 1.5%) the difference should be obvious between 360 and 300 bp. Run beside a 100bl ladder and/or use control of the empty vector.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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

Post by JackBean » Thu Mar 24, 2011 6:19 am

you have 300 bp vector? =-O
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

Post Reply

Who is online

Users browsing this forum: No registered users and 2 guests