Science Buddies: "Ask an Expert"

For a project that involved protein manipulation, I was hoping to use a oligonucleotide-directed mutagenesis. I was wondering about the limitations about this technique, esp. on the length of the oligonucleotide primer

Hi,
I am checking on this for you today. Are you intending to use any specific kit on the market for site directed mutagenesis? Or do you have a protocl you intend to use that you can share with me?

Thanks,
Tonya

Hi,

I looked into this a little more today and found this site, which offers a pretty good overview of the technique with more links at the bottom. http://escience.ws/b572/L4/L4.htm

One example of a kit you could use can handle a maximum insertion of 21 nucleotides, delivered in a plasmid of 2-8kb. This is just one example, as many companies market similar kits that will have their own recommendations for use. http://tools.invitrogen.com/content/sfs ... ns_bro.pdf this brochure does explain some limitation of general protocols and how the kit overcomes those limitations.

As I said in my post this morning, if you have a protocol you intend to use, you can post it here and I will look into the details with you.

Tonya

Hi- Just to follow up on Tonya's reply, I'm actually doing a ton of PCR-mediated mutagenesis at the moment. The kit that I've had the best luck with is the QuickChange kit from Promega/Agilent
(http://www.genomics.agilent.com/Collect ... PageID=383).
They have a ton of different kits that vary based on how long your plasmid is and adding several mutations in the same reaction - if you design your primers right (they have a primer design tool on their site - you need to set up an account first:
http://www.genomics.agilent.com/Collect ... &PageID=15) and have them purified (by HPLC or PAGE) this will work like 99% of the time. (you can use their primer design tool with any PCR-mediated mutagenesis kit, it's just a useful tool ). It's a few extra $$ to have them purified, but it makes a big difference in efficiency of the reaction. It's important to have your primers purified because in the normal oligo synthesis process there is a certain percentage of oligos that are truncated early, meaning you'll wind up with a mix of the primer that you want with some degenerate primers in there. These mutagenesis primers are usually long, about 30-60 bases, so if you have your mutation right in the middle of the sequence, and you have some degenerate primers, you'll wind up with some primers that don't have the mutation sequence. Then the polymerase has a chance of just amplifying off the primers without the mutation and you'll wind up with a non-mutated plasmid.

Hope that helps a little!
Stephanie