Promega :Mutagenesis Systems - (Feb/12/2007 )
Hi All,
Does anyone have experience with Promega's Mutagenesis systems : GeneEditor/Altered sites, I would like to know about it.
My problems is- I need to mutate three bases on my plasmid (a single amino acid in my protein). So if anybody has used these previously please describe
1). the advantages of one over the other
2). which is more suited for my problem
3). the mutation size limit in terms of bases that is possibile with these kits, and yes
4). any precautions/tricks.
Thanks for the help in advance!!
Could you explain what you are attempting to do?
Most mutagenesis and things in biology do not require a kit, merely knowledge.
-Matt
Thanks Mat for your reply
I am trying to mutate the methionine to Lysine/Arginine in my protein for trypsin cleavage. Since I need to cleave off the protein from the peptide of interest. The cleavage at methionine with CNBr is not efficient.
Hope this helps
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Could you explain what you are attempting to do?
Most mutagenesis and things in biology do not require a kit, merely knowledge.
-Matt
[/quote]
No kits required whatsoever. PCR mutagenesis. This is the best image (right hand image) I can find at the moment but have a look and see if you can figure out what's going on. Really easy to do once you know what's happening.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.568
P.S. How do you stop those stupid smiley faces coming up whenever you put the letter B next to a bracket?
Hi killerkoz17,
Thanks for the reply.
One last question, what is the largest mutation in terms of size (bp) which can be performed?
Do you have any past experience??
Thanks
Do you have any past experience??
Hmmm, good question. Well the mutation has to be designed in the middle of the primers for starters and I think for small mutations of 1-2 bp you can probably get away with designing a high Tm primer around 70C. For bigger mutations however, it's really a matter of trying to get enough bases on each side of the mutation so that the sequence on each side will have a high enough Tm to anneal during the initial PCRs and the joining PCR. In these instances, both sides of the primers need to be treated like they are two primers within the one and must have sufficient Tm by themselves (as though each side is participating in its own PCR) or else either the initial PCR or the joining PCR will not work. I can't see anything wrong with having a large number of mutated bases, say around 10, in the middle of the primers, as long as the Tms on both sides of the mutation are sufficiently high. Obviously where possible any native sequence you can keep (within the sequence you wish to mutate) is welcome. It's really a matter of seeing how much you can get away with if you want to perform a lot of mutations. The longer the primers/higher the Tms on each side of the mutation, the more mutations you can include and the more likely you will be successful. Below are some notes I took from this link a while ago.
http://www.promega.com/tbs/tm001/tm001.pdf
-single-base mutations
-17-20-base oligonucleotide with the mismatch located in the center
-8-10 perfectly matched nucleotides on either side of the mismatch
-two or more mismatches
-25+ base oligonucleotides or longer are
-12-15 perfectly matched nucleotides on either side of the mismatch.
-four-base insertions and deletions
-26 and 27 bases total
-larger deletions require
-20-30 matched bases on either side of the mismatched region.
Btw, here is another link on how to mutate your sequence. It's different to the standard protocol and takes a bit of thinking to get your head around it but it's a cracker idea.
http://www.biochem.ucl.ac.uk/bsm/nmr/proto...utagenesis.html
Seeya, Rob