site directed mutagenesis - (Apr/07/2005 )

I am doing site directed mutagenesis using stratagene quick change XL site directed mutagenesis kit. Can someone explain me the role of 2-mercaptoethanol in this process. Also I would like to know if NZY+ broth can be subsituted by LB.
thanks

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Hi,

I don't exactly know what the mercaptoethanol does to the cells (Stratagene claims it would increase the compency of the cells). To your other question: yes you can use LB instead of NYZ+ but you have to keep in mind that you will work with special E.coli cells. NYZ-medium is more suitable to grow up your cells after the heat-pulse.

Good luck for your PCR!
Freiberger

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thnx freiberger

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Hey Molbioguy,
just to let you know (if you're doing an awful lot of these reactions and want to save a bit on consummables!) we don't bother buying the kit, just the Pfu polymerase. You can just do the PCR, digest 1 ug with Dpn 1 (to remove parent DNA), purify and transform. We used LB but I think as long as the media is suitable for the bugs youre using it should be ok!

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i am trying to do quikchange SDM for quite sometime now, but it doesnt seem to work. i want to insert restriction sites in my gene. can someone tell me if the primers for this have to be designed just as given in the manufacturer's manual (stratagene) or there is some modification to be done to the primer designing part? and also if i might have missed out on anything which might not be there in the manual...

thank you

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For rapid and cost-effective method, I strongly recommend recently published method using type IIs enzyme (Ko, JK and Ma J, Am J Physiol Cell Physiol. 2005, 288: C1273-8.). This method originally published by Tomic M (Nucleic Acid Res. 1990, 18: 1656.). My colleagues and I also successfully generated various mutants (substitution, insertion, deletion and chimeragenesis) by this method. You need only four primers (just desalted not purified, it’s very cheap!) and type IIs restriction enzyme. The mutagenesis efficiency is close to 100%.

Presently, the overlap extension method, megaprimer method, Quick Change method (Stratagene) are prevalent among numerous PCR-based mutagenesis methods developed commercially or non-commercially. However, all of them has shortcoming for applying to the diverse mutagenesis strategies including base substitution, deletion, insertion, chimeric gene generation and multiple-site mutagenesis. Especially, they show low mutagenesis efficiency (below 40%) for genes with long sequence (>3 kb), GC-rich region, tight secondary structure, or tandem repeats and for a long frame mutation (> 10 bp).

If you have plan to use commercial kits, another choice for short way for your research is use of mutagenesis service company. I realized the mutagenesis cost using commercial kit is never cheap! My estimate is $200 ~ 250 per one mutagenesis reaction using kit (one kit rexn., $20 + purified two primers, $150 + three clone sequencing, $30 + subcloning reagent, $30 : SM, media, agar plate, buffer, tube, tips --- and time). Besides, purchasing kit for just one or two mutant generation is money wasting because remaining kit reagents are useless. Recent biotech companies provide rapid and precise mutagenesis service in affordable prices. Some people in our lab use Mutagenex Inc. (USA) and I found other companies offering in low price:
Mutagenex: $249 per mutation, USA
Topgenetech: $269 per mutation, Canada
MCLab: $280, USA
You can also find more other companies that have different technology and service criteria.

In conclusion, my recommendation is,
Efficient and cheap method: Type IIs method!
Easy way but need money: company rather than kit!
For more discussion, you can contact to choik1@umdnj.edu

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