QuikChange® Site-Directed Mutagenesis Kit

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Bouchra
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QuikChange® Site-Directed Mutagenesis Kit

Post by Bouchra » Thu Sep 06, 2007 11:20 pm

Hi,

I was looking for a fast way to introduce a direct mutation in a gene, so I checked the manual for the QuikChange® Site-Directed Mutagenesis Kit from Stratgene. I would like to now if some one has used this kit, if so, did you use your own plasmid to clone your gene of interest or did you use the pWhitescript plasmid provided in the kit?

Thanks

Bouchra

blcr11
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Post by blcr11 » Fri Sep 07, 2007 2:08 am

It works with any plasmid. You just need to be mindful of how big your plasmid is and adjust your pcr extension times to make sure you give the enzyme enough time to read through the entire plasmid + gene (not just the insert itself). For the Hi Fidelity enzymes I use this usually works out to around 6-7 minutes of extenstion time per step, which is probably a little longer than is absolutely necessary, but I prefer to err in that direction.

ShineGene
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Post by ShineGene » Sun Sep 23, 2007 2:12 am

that is very good.i have used it.
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http://www.synthesisgene.com

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Ammie
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Post by Ammie » Tue Oct 02, 2007 3:02 pm

Not really a answer to your question but...

I have used Multi QuikChange kit from Stratagene where you can mutate up to 5 nt's on the same time.

When I sequenced 3 colonies, two of them had 3 of 5 mutations and one hade 4 of 5 mutations. I will now sequence 3 more colonies and hope to find one that have them all.

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