:: Search forums :: Top Users :: Reagent
Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
|
Re: Site directed mutagenesis with PCR
Posted by: charan (IP Hidden, Junior member, 14)
Date: September 27, 2005 03:57PM
Hi
I am having issues with quick change mutagenesis kit. I am trying to mutagenize a gene ,introduce one base substitution in a 2.5kb gene cloned into puc19. my oligos are 37base long with 18 base homology on either side I follow exactly the same procedure as per the manual but only variation is that a) my oligos are not PAGE purified b) I do not use the competent cells that come with the kit My issue is that I get NO colonies at all I tried both chemical transformation and electroporation. But nothing seems to work. IO saw someone with similar issues . My extension time is 1.5 min/kb and final extension is 10 min. Can you suggest something. Also I have no other option but plasmid mutagenesis. So has any one had any experience with other kits???????????/ Thanx
Re: Site directed mutagenesis with PCR
Posted by: besttuti (IP Hidden, New member, 5)
Date: May 6, 2006 11:21PM
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
|
We are moving ... Please post to our new community forums
