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Invisiblemicro
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Site-Directed Mutagenesis
    #2033481 - 10/22/03 07:56 PM (14 years, 6 months ago)

O.k. -- I have to change the genome of a plasmid because one of the sites that's used for antibody production is in two different places. My boss wants me to change a G to a C in the beginning of the site (a transposon.) There's this kit, apparently, to do it, and the instructions seem pretty straight-forward, but how the fuck does this work?

It uses a thermocycler and the number of cycles, apparently, determines the type of mutations....

I looked it up and couldn't find anything. Thanks,

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OfflineMetaShroom
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Re: Site-Directed Mutagenesis [Re: micro]
    #2035740 - 10/23/03 12:22 PM (14 years, 6 months ago)

The detail would depend on which kit you are using but basically you need two primers containing the mutation which are complimentary to the sections of plasmid either side of your mutation site. These are then extended by a polymerase leaving nicks and the original DNA is digested by something like DpnI which only targets methylated DNA. The nicks are repaired when you later tranform the mutated plasmid.


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Invisiblemicro
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Re: Site-Directed Mutagenesis [Re: MetaShroom]
    #2035779 - 10/23/03 12:38 PM (14 years, 6 months ago)

Thanks -- that makes sense :wink:

BTW -- it's something like the E-Z QuickChange kit made be some company like stargene or something -- don't know exactly off the top of my head and I'm not in that lab.

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Edited by micro (10/23/03 03:44 PM)


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