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Query regarding primers for quick change mutagenesis

Problem in primers

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3 replies to this topic

#1 kartiga natarajan

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Posted 26 March 2013 - 09:00 PM

Dear All,

I have designed the primers for quick change mutagenesis in agilent software....I checked the heterodimer, homodimer and hairpin in one oligo analyzer...http://eu.idtdna.com/analyzer/applications/oligoanalyzer/. In that i found out that the primers had high energy to form homo and heterodimers (< -3 kcal/mole) i.e. both my primers have -9 kcal/mole and -6 kcal/mole energy. So, i looked for better primers, by reducing the bps but it too resulted similarly. Now what shall i do? Already the primers which i used did not work so im designing new one...So plz help me out in getting good primers.Also comment on the primers i have designed...Waiting for your reply...Plz do reply soonAttached File  Agilent new primers (1).pdf   245.39KB   211 downloads

Attached Files



#2 kartiga natarajan

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Posted 26 March 2013 - 09:02 PM

These are the results obtained for 353D primers in oligo analyzerAttached File  353D primers.zip   1.51MB   72 downloads

#3 bob1

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Posted 27 March 2013 - 01:34 AM

Typically for quikchange you use primers that are completely complementary to each other, so I would expect there to be a lot of heterodimerization. You need to optimise the annealing temperature quite well for this protocol to work.

Another option is to use primers that don't completely overlap...

#4 HOYAJM

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Posted 01 April 2013 - 12:14 PM

I don't think -9 kcal/mole is terrible. I usually shoot to keep everything under -9 kcal/mole for all of my primers. bob1 is right that they are completely complementary so they will obviously form dimers. But, you need to remember you are not trying to amplify for purification where you need tons of product. You just need enough to transform into a few bacteria so you get colonies. Thus, I use 50C for annealing ensuring I will get the right product somewhere, even though there will be dimers and non-specific priming.

Agilent even states that if you run your PCR out on a gel, you may not even see a product, but transform anyways, because it should still work. It just doesnt seem right, but I can attest that "no product" reactions can still have the mutation.




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