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Site directed mutagenesis problem

Salam all,

 
One more molecular biology problematic situation: I am trying to do site directed mutagenesis for one of my vectors to create a restriction site I can use for my cloning to change a U6 promoter.

 
This vector is an AAV-transfer vector: it has 2 ITR's, CMV promoter, EGFP hGH Poly A, AmpR, f1 ori and ColE1 ori. A part from the ITR's it's a classical vector (it's the main issue!!!) (map attached, ITR's are the little red sequences)

 
Here's the problem: When I did my mutation using quick change kit from stratagene no colonies were obtained after transformation. I concentrated the PCR product by precipitation and after transformation I got some clones but none of them was mutated when screened with the enzyme I was trying to introduce. Of course I have a positive control, a pBS vector was easily mutated and the restriction site was introduced in a single step. The enzyme is working, the Taq is working too. All the controls were performed and all the components seem Ok.

 
The very strange point is: I tried in the past to mutate a cDNA that was cloned in the same backbone vector. The same problem: No way to do it. Fortunately, I had the cDNA in a different vector, believe or not the mutations were introduced since the first round with the same primers. I just cut and paste it in my viral vector.

 
My question is: as the only difference between this viral vector and other one is the IRT presence, can you imagine that this sequence my interfere with the PCR reaction when mutating?
I'm trying to develop cell specific conditional shRNA knock down that people can use with Cre transgenic mice, and modifying my AAV vector is the limiting step.

 
I will be more than grateful if you guys can help with this.
Thanks in advance and best regards

 
 
 

___________________________________________
Amine BAHI, PhD.
Yale University School of Medicine

Salam Hassan, Thanks a lot

Salam Hassan,

Thanks a lot for your input: my primers are on the U6 promoter sequence. On the map it's between SfiI ans SapI restriction sites.

Regards

 

Amine you didn't answer the

Amine you didn't answer the 2nd question, but assume you have a good PCR product, in this case it's not a vector problem, may be a problem in the primer (bear in mind sometimes primers have a wrong sequence, I would add this may be rare anyway), does your primer need to be phosphorylated (5 prime), if yes, are you sure it is.

At the end I will add that if you really can't do it this way, there is a way that will work almost 100%.

Buy the Kit from Promega pALTER mutegenesis KIT :  Altered Sites II in vitro Mutagenesis Systems

In my hands this was the best and more sure way of doing site-directed mutagenesis, I was able sometime to introduce as much as 3-5 mutagenesis primers in one single round. But you have to use the ssDNA protocol.

Let me know if you still have a problem or question

 

Hassan BADRANE Pittsburgh, PA

Promega Approach

 Thanks a lot Hassan for your suggestion. I don't really mind to buy this kit from Promega but my question is: what's the difference compared with the one from Stratagene?? how ssDNA protocol will improve mutation efficiency??

Regards

what you have to do is to get

what you have to do is to get the fragment you want to mutagenise (better if it's ~ 0.5 - 1 kb) out of your vector using unique RE sites, gel purify both bands. Then clone into pALTER (Promega), prepar ssDNA then do mutagenesis reaction. In a week you should get your mutagenized fragment that you can put back in the original vector. Best if you look at the manual of the kit (link provided in the previous comment) before doing anything to insure compatibility in your case.

Let me know if you need more details.

Hassan BADRANE Pittsburgh, PA

Amine, could you please

Amine, could you please specify:

where your primer anneal?

Do you have a good PCR product ?

Hassan B1

 

Hassan BADRANE Pittsburgh, PA