Login

Join for Free!
118319 members


ligation rxn on gel was a smear

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

Moderator: BioTeam

ligation rxn on gel was a smear

Postby dakims » Wed Jan 20, 2010 6:32 pm

i ran a ligation reaction on a gel and got a very faint smear. the smear was so faint i may even be wrong. i loaded 5ul from my ligation which had .1ug dna total. I'm really not sure what happened. I ran a control with everything but t4 ligase and i got two bands. so contaminant can't be in the buffer or water or dna sample. so something had to be wrong the the ligase? i dont have undigested vector (i sap'd it all) to run it against so i'm not sure what to do! i went ahead with the transformation praying that it will work. but i think i should run another ligation. any suggestions?!
dakims
Garter
Garter
 
Posts: 18
Joined: Mon Nov 26, 2007 5:21 am

Postby JackBean » Wed Jan 27, 2010 7:49 am

Yeah, you probably will have to try another ligation. Best try some other ligase.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Re: ligation rxn on gel was a smear

Postby clevermizo » Thu Jan 28, 2010 4:03 pm

With that much DNA in there you probably have a lot of incorrect products. Typically I use <100 ng total DNA (i.e., 50 ng vector and 3:1 molar excess of insert). I can't really recommend using 1 ug of DNA in a ligation reaction. Now, if you use as small of an amount of DNA as I do, you won't likely be able to resolve your ligation products easily on a gel (I dunno, maybe you can). But I just transform and screen colonies. Anyway, this seems to work out. This month I had to make 15 constructs, so hopefully I know whereof I speak.

Of course I don't know all your reaction conditions, amounts of vector/insert, units of ligase, reaction time, etc. You mind sharing that?
clevermizo
Garter
Garter
 
Posts: 7
Joined: Thu Jan 28, 2010 3:48 pm



Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests