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mutagenesis by PCR or just use a kit


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5 replies to this topic

#1 wizzkid

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Posted 08 March 2010 - 02:00 PM

what would be the advantages of uses PCR to intoduce a mutation over using a site-directed mutagenesis kit like Stratagene? I would like to have a few ideas before a lab meeting with supervisor soon. i know using PCR is more cost effective however the kit is quicker and easier to detect if your mutation has been introduced.
would appreciate the help from all the molecular biology buffs! :P

#2 Rsm

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Posted 08 March 2010 - 06:29 PM

Hi,

I don't really understand what you mean with "PCR", because the Quick Change Kit uses PCR as well. If you mean overlap extension PCR for introducing mutations, that would take 2hrs longer, agreed. But at least you'll know that your vector backbone is ok, and not accidentally mutated... Cloning efficiency is also higher, and you're more likely to really get your mutation. Primer design is not as tricky, and you don't need to phosphorylate / HPLC purify them.
I don't think that it is easier to detect mutations using the kit. Of course, you have the control reaction, but that does not mean that you're mutation is present in your plasmid. The positive control is optimized, your mutation is not.

Best,
Minna
I got soul, but I'm not a soldier

#3 Stuck with Ligation

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Posted 24 March 2010 - 11:31 AM

Overlap extension! I do not like the quickchange kits because you cannot see the product on a gel. I like the overlap extension methods because the product can be seen.

In my experience, I have had lots of quickchanges fail and I have not been able to determine if it was the PCR or the restriction step; however, when I do overlap extension it works almost every time.

#4 Dukey

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Posted 24 March 2010 - 03:22 PM

Overlap extension! I do not like the quickchange kits because you cannot see the product on a gel. I like the overlap extension methods because the product can be seen.

In my experience, I have had lots of quickchanges fail and I have not been able to determine if it was the PCR or the restriction step; however, when I do overlap extension it works almost every time.


I have never had a quickchange fail, the kits work very nicely in my hands! It all depends how good your primer design is quite honestly.

#5 DocFlow

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Posted 25 March 2010 - 06:17 AM

I've used both methods and quikchange is by far my favorite. you won't have to order kits. Just design your primers, buy that pfu turbo polymerase and purchase DpnI from NEB or another supplier. works just fine. Use your regular competent cells too and you'll easily get your mutation done without all that gel fragment isolation and subcloning. As an advise, after you've identified positive colonies subclone a smaller mutation containing fragment into a new "clean" plasmid so that you won't have to sequence over a long stretch of nucleotides.

#6 jiajia1987

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Posted 25 March 2010 - 04:51 PM

Overlap extension! I do not like the quickchange kits because you cannot see the product on a gel. I like the overlap extension methods because the product can be seen.

In my experience, I have had lots of quickchanges fail and I have not been able to determine if it was the PCR or the restriction step; however, when I do overlap extension it works almost every time.


I have never had a quickchange fail, the kits work very nicely in my hands! It all depends how good your primer design is quite honestly.


I go with Dukey. I have never had a problem with quikchanges unless my primer design is bad. When the primer design gets bad, you can get tandem inserts of your primers!




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