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Site-directed mutagenesis: maximum substitution


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#1 HOYAJM

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Posted 31 March 2014 - 07:00 AM

Hi, I would like to change a 5 amino acid (max 15nt) sequence in a 11Kb plasmid. What is the maximum substitution you can successfully complete with one reaction? I have done extensive SDM before, but only for 1-3 nucleotides. Here I will be changing nucleotides throughout a 15 nucleotide sequence to change the coding sequence of 5 sequential amino acids. I was thinking of using the NEB Q5 site-directed mutagenesis kit.

 

Will these kits work for substitutions this large? I was going to use 20-25 nucleotides of complementary sequence downstream of the mutation for the 3' anchor.

 

Please let me know if you have had experience with substitutions of this size in a single reaction.

 

Thanks



#2 bob1

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Posted 31 March 2014 - 12:03 PM

I don't have experience with large ones, but I thought the limitation was related to the size of the mutation - for instance if you wanted to change all the bases in a 15 nt site, that would probably be fine, but much more and it would start to get a bit "floppy" and you could get some re-annealing in the mismatched regions.  Changing a few of the bases in a 15 nt sequence should be fine I think.






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