isolation of DNA from a plasmid for site-directed mutagenesis? - do researchers actually do this? (Jan/01/2006 )

hello, happy new year!

I would like to make a mutant protein- we collaborate with another lab that has made a plasmid with my desired protein's gene. I would like to know if I can perform site-directed mutagenesis on this DNA from the plasmid... in other words, can this plasmid DNA serve as my DNA template?

I want to use (transform) the exact plasmid that is already made by this collaborating lab, except that I want to perform site-directed mutagenesis on the gene that is in it.

I imagine I would:
1. cut out the gene + flanking regions from the plasmid
2. amplify this gene fragment using primers with my specific mutations
3. clean up the pcr product
4. ligate the pcr product back into the original plasmid (?). Or should i instead ask them for the parental plasmid that they made their plasmid from?

I haven't cloned for many years, and I would appreciate any suggestions you might have!

Much luck in 2006

If your plasmid isn't too big, you can do SDM on the plasmid itself. Check out stratagene's quickchange kits (www.stratagene.com). (you don't have to buy the kit, you can easily perform it by yourself with cheaper enzymes and so on).

Just PCR amplify the entire plasmid with primers containing the desired changes.

How many changes/types of changes would you like to make? That will effect how many primers you use and the protocol.

-Matt

I want to use (transform) the exact plasmid that is already made by this collaborating lab, except that I want to perform site-directed mutagenesis on the gene that is in it.

I imagine I would:
1. cut out the gene + flanking regions from the plasmid
2. amplify this gene fragment using primers with my specific mutations
3. clean up the pcr product
4. ligate the pcr product back into the original plasmid (?). Or should i instead ask them for the parental plasmid that they made their plasmid from?

hi dear
you can directly amplify with your primers dont worry first chek the frame and orientation of plasmid and restriction site then you can go easliy in same vector or another vector i think it will better if you use a plasmid with more than on mcs sites see the website of invitrogen.

hi dear
you can directly amplify with your primers dont worry first chek the frame and orientation of plasmid and restriction site then you can go easliy in same vector or another vector i think it will better if you use a plasmid with more than on mcs sites see the website of invitrogen.

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 have 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 have shortcomings 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). Ko’s method has been very successful for every situations mentioned above.

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: Ko’s Type IIs method!
* Easy way but need money: company rather than kit!

For more discussion, you can contact to choik1@umdnj.edu