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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Site directed mutagenesis
Posted by: nithya (IP Hidden, New member, 3)
Date: January 19, 2006 10:16AM
I am trying to change three consecutive bp in a approx 5 kb plasmid using the stratagene kit. The PCR appears to be happening and after Dpn1 digestion, I transform the product into DH10B cells. I also get a lot of colonies on the plate. Now, the problem is that upon sequencing, none of the colonies (upto 5 in each case) seem to have the mutated sequence. All of them have the same wild type sequence. Does anybody know why this is happening?
There is a poly G sequence right next to the site in question..could this be a problem? If so how can I improve the design my primer? Is there some other method to obtain this kind of mutant? Thanx
Re: Site directed mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: January 19, 2006 12:47PM
Are you using the Dpn from the kit? If so is it still active.?
You might want to check this by digesting a control DNA with this DpnI. Also try running a control reaction without polymerase which after digestion should give you nothing upon transformation. Hope this helps.
Re: Site directed mutagenesis
Posted by: nithya (IP Hidden, New member, 3)
Date: January 19, 2006 04:43PM
I am using Dpn1 bought separately. Out of the five different dugested PCR products transformed, only two showed 100s of coloies. So, I am not sure it is the Dpn1 causing the problem.
Re: Site directed mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: January 19, 2006 06:29PM
What are the three different plasmids/digests? Are these different primers? What is the resistance of your plasmid?Is there any chance that your competent cells having the same resistance?
Re: Site directed mutagenesis
Posted by: nithya (IP Hidden, New member, 3)
Date: January 20, 2006 09:58AM
I am working on three different mutations, using three pairs of primers in three different PCR reactions.
My plasmid has amp and chloramphinicol resistance... while the comp. cells do not have both.. colonies grew on the amp plate while it was significantly less in the amp+cmp. plate...
Re: Site directed mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: January 21, 2006 08:45PM
Amp tends to get degraded fast on plates So U R results kind of make sense. the amp must have got degraded which explains the number of colonies.
We have had a lot of trouble recently with one particular construct. We got around by first optimizing the PCR by increasing the elongation time,making sure that it works and then transforming into FRESHLY prepared competent cells (chem and electrocompetent) and plating on FRESH plates. I am still curious about your cells how do you make them competent?
Re: Site directed mutagenesis
Posted by: besttuti (IP Hidden, New member, 5)
Date: May 6, 2006 12:42PM
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, please contact to choik1@umdnj.edu
Re: Site directed mutagenesis
Posted by: huangz123 (IP Hidden, Junior member, 19)
Date: May 6, 2006 09:06PM
Thanks for the valuable information. I would definetly have a try on Ko's method. Some commercially mutagenesis kits are ridiculously expensive though they are convenient.
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