Induce mutation in my gene - (Jul/05/2007 )

Hi, I am graduate student starting out fresh. I have very little exposure to work with cloning.

I have a gene in a Gateway entry vector. I want to induce a mutation in this gene. How would I do this. Please explain as simply as possible

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You need to get primers with the mutation and amplify the plasmid.

Look for info in companies which supplies site-directed mutagenesis kits. They have all the info and protocols and programs which can help design the primers with the desired mutations.

Good Luck !!!

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for mutation in a gene site-directed mutagenesis is not only the way i think............i am not sure for graduate student what is the practice to do mutaiton in a gene.........you can ask you PI about the method only , then explore the method to use properly

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yeah, you could use Taq with Mn ions, to make error prone extention. Adding ITP (inosine) also helps increase mutation rates.
You could also use mutagenic polymerase. They are even ones being sold which don't have that much transversion bias. You could also use fragments of related genes acting as primers to introduce new domains, or larger sequence variation.

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Thanks everyone! Theoretically I understand what you guys are saying. I read up some stuff. But I don't think my lab has the capacity to buy kits, so I have look for something cheap. Now while I understand theoretically what would happen, how would I make primers: do you have a formula if I was getting my own primers made and use the other material from what our lab has?

My boss wants me to explore temperature sensitive site directed mutagenesis for my mutant. I have no clue how to do this. Can anyone give me clues???

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You could use the programs (stratagene's website) to design primers and then use stuff in the lab to generate the mutants.

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