inserted extra fragment after PCR reaction

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

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
fred
Garter
Garter
Posts: 1
Joined: Wed Mar 22, 2006 4:07 am

inserted extra fragment after PCR reaction

Post by fred » Wed Mar 22, 2006 4:22 am

hi, every one,

I did a site-directed mutagenesis of plasmid using Stratagen kit. The sequencing showed there is an inserted fragment that is almost the primer except the AAC as follows: "catccgctt tgtttCcaac tcatgggAACcatccgctt tgtttCcaac tcatggg"

The primer sequence is Forward:5'catccgctt tgtttCcaac tcatggg3'

Should I increase the annealing temperature to solve this problem?

Yours sincerely,

Fred

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

Post by canalon » Wed Mar 22, 2006 4:49 am

What you did is taht you made a PCR with a plasmid taht did not fully match your starting sequence and then inserted in the plasmid. Am I correct?

I am not sure that changing PCR conditions would change a lot of things. You are in the middle of the primer, and an insertion at this site during PCR seems unlikely.

I would rather think of the digestion/ligation process in this case. How did you inserted it in the plasmid? where was the restriction site?
Patrick

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

User avatar
LilKim
Coral
Coral
Posts: 436
Joined: Mon Mar 20, 2006 8:36 pm
Contact:

Post by LilKim » Wed Mar 22, 2006 2:17 pm

If i were to guess.. I would say decrease the anneaing temp.Thereby decreasing the stringency of the PCR rxn... as a result the primers will not prime as specifically as they used to... and this will allow the primers to prime in the presence of the mismatch.

I've heard that you'd have to decrease the temp. quite a bit if you have mismatched-primers. But, i've never done it so ... i can't give you a ball park figure.

When you check your rxn, You'll probably end up with a ton of bands on your gel .. Look for "your" band .... and then run-out the entire PCR rxn for a long time (You'll have a LUMP of DNA so you'll need to run it out to get better resolution and a sharp-er band). .. at that point you can cut-out the band from the gel .. and gel-purify with a kit.

I dunno if you work in a research lab ... but I'm sure that you can find someone in your lab ... or a lab down the hall. Someone will be able to help you ... cuz the problem that you're having probably isn't too difficult to trouble shoot (if you find someone who really knows what they're doing)

I, on the other hand .. have a clue, but I don't really know what i'm doing ...

if all fails go back and CHANGE!
1. dH20
2. Taq pol (Hi Fi Taq)

Also make sure that you're using enough MgCl2 (MgSO4)

hope things work out ... good luck!

- Kim

Post Reply

Who is online

Users browsing this forum: No registered users and 6 guests