[RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

From: Fred
Date: 3 February 2012 17:13


Dear CCP4users biologists,
I'm trying to make a single aa mutant of a 5.7 kb non commercial vector with the Agilent's Quick Change Site-Directed Mutagenesis Kit. I have strictly followed the instructions manual, however, I could not be able to transform bacterial cells with my PCR product. I can observe the amplified PCR product before and after DpnI digestion (see image in http://ompldr.org/vY2x3aw), but cannot get any colony on LB plates. I'm using very fresh super competent cells so that I've got dozens of colonies with 60 ng of the parental/non-mutated vector as positive control. The bands in the referenced image corresponds to 2.5 microL of a 50 microL reaction volume. I usually concentrate it to 4 microL before transformation. Also, I've already optimized the primer's temperature annealing (best is 62 oC) and I've increased the extension time up to 9 min. Is there anything else I can try?
Any help is appreciated!
Regards.
Fred

P.S.: Agilent's e-mail support is not working.
P.P.S.: this might not be of other's interest, address the answers, please, to my e-mail only.

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From: Raji Edayathumangalam


Hi Fred,

If I understand you correctly, you are concentrating your 50uL reaction to 4uL and then transforming all of it. If that is correct, that is WAY too much DNA, especially based on what I see on your gel. Too much DNA inihibits transformations and that is a very common mistake. Assuming I am reading you correctly.

Simply transform say 2.5uL of the PCR reaction (post DpnI) directly into cells and that is likely to work. You could also try, say a bunch of transformations with 1uL, 2.5uL and 5uL.

Good luck and I can help you out further to get your mutants!

Raji

--
Raji Edayathumangalam

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From: Artem Evdokimov


Hi,

1) anneal your PCR product (after dpn)
2) use smaller amount of material to transform, as already pointed out
3) if you could share the primer design, maybe we can find something in there

Artem

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From: Dima Klenchin


Sounds like you don't have competent enough cells. 60 ng should give millions, not dozens of colonies. (Decently competent cells are 10^5/ng of plasmid). Only a tiny proportion of the observed polymerase product in QuickChange reactions is actually transforming, so you have to assume that you start with a lot less than 1 ng. So if you are not seeing at least a 1,000 of colonies after 1 ng plasmid transformation, chances that you will have positive clones in your QCh transformation are pretty low.

- Dima

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From: Fred


Hi CCP4 list,
Thanks everyone who have answered my post concerning to mutagenesis.
From quick reading most of the answers, the following seems to be a consensus:
1) Do not concentrate your PCR product;
2) Too much DNA and/or impurities like salts or whatever can inhibits transformation;
3) Purify your PCR product before transformation if possible or use 3 of 4 microL of it. This is more or less the amount of DNA showed in the uploaded image.
Kind regards,
Fred

P.S.: I'll let you know the results.

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From: Nian Huang


Just a reminder. Quickchange is not PCR. It is linear amplification. It is very hard to see a band in the gel if you follow the standard protocol.

Nian

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From: Roger Rowlett

We prefer to do MEGAWHOP PCR and use 1-5 uL of the DPN digested PCR product. This is extremely reliable with commercial competent cells.  See http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Recombinant+DNA+Protocols#Site_directed_mutagenesis_by_Megaprimer_Quick_Change_MEGAWHOP_PCR for details. Allow 1 min/KB each cycle for whole plasmid PCR extension.

Roger Rowlett

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From: Artem Evdokimov


Quickchange is obsolete :)

Artem

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From: Fred <


Hi CCP4 list,
Thank you very much for additional messages and references.
Here goes the image of the "PCR" product before digested and after digested and cleaned.
 http://ompldr.org/vY29jbA
The results of the transformation of 3 microL (90 ng) of non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 digested sample gave two colonies only; and transformation of 3 microL(90 ng) of cleaned product gave 14 colonies.
So, if the amplification is not abundant, chances are that home made competent cell will not be transformed with the digested product. Don't want start another discussion but, is there any reason for differences in the transformation efficiency between the parental and the mutated (cleaned) plasmids?
All the Best,
Fred

Em 03-02-2012 16:14, Fred escreveu:

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From: Xiaodi Yu

Hi Fred:

For the mutated plasmids, it generates the nicked dna, so the transform efficiency will be lower compared to the parental plasmids. And that is the reason why people usually use super competent cell to transform these nicked plasmid. It seems ok to me to get 2 or 14 colonies from the nicked plasmid. You just need ONE. And usually for these mutagenesis PCR, the success rate is pretty high. I suggest you to sequence these colonies. And you can get it.

Yu Xiaodi