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Single codon insert

cloning mutagenesis

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#1 ostiaziocan

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Posted 01 December 2013 - 12:33 AM

Hello everyone,

 

what do you think is the easiest and fastest way to insert a single codon in a coding sequence cloned into a plasmid? I was wondering whether site directed mutagenesis might work...

 

Thanks smile.png

 

 

 

 



#2 bob1

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Posted 01 December 2013 - 11:30 AM

There are basically 2 ways to do this - both will probably take about the same time (2-4 weeks), but one costs significantly more than the other.

 

You can either get the gene with the mutation synthesized by a company and then subclone into vector of choice ($$$ depending on sequence length), but requires little input of time from you; or you could do SDM, which is relatively cheap (you don't need to buy a kit, as you can buy all the necessary components yourself), but can take some time, and is quite hands-on.



#3 phage434

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Posted 01 December 2013 - 03:02 PM

You can do traditional site directed mutagenesis (such as Quickchange, or a home brew version of it). You can also use PCR of the entire plasmid with primers containing an offset cutter restriction site. Purifying and cutting with the RE, followed by ligation will also give the product you want.



#4 ostiaziocan

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Posted 01 December 2013 - 08:58 PM

Wow, thanks for the quick reply and suggestions! smile.png

 

Indeed, my initial idea was to go for SDM... I have used it to insert an HA-tag (27bp) but I was not sure it would work to insert just 3bp.  

I thought that the need of creating a 3 bp hairpin would not allow the primer to bind the dna template.

I will go for SDM ph34r.png ,

Thanks again







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