PCR-Based Mutagenesis DNA Template?

Question

I've created mutagenic primers from the cDNA sequence of a gene of interest. I'm having trouble with the PCR in that I'm not getting any bands of amplication. I'm using a genomic DNA sample as the template DNA and am wondering if I shouldn't use a plasmid DNA for the DNA template - any suggestions? Thanks!

Answer 1

There are too many things that might have gone wrong.
You are saying your template is a genomic DNA and then that it is plasmid ("if I shouldn't use a plasmid DNA") ? It can't be both, unless you mean you have a genomic library in a plasmid...

plasmid templates are very good for PCR so that is definitely not the problem.(Unless the plasmid preparation is not good and you have e.g some ethanol or isopropanol or whatever in it.)

If you meant that you have a choice between genomic and plasmid template go for the plasmid
You have to check again the design of your primers, the putative Tm, the size of the DNA so that you let elongation occur for enough time, etc...

Some polymerases cannot cope with very long and difficult templates under usual conditions so you might have play with the conditions or change polymerase or use a polymerase mix.

We don't have enough info to be more specific. You just have to go through the design again carefully to see if there are any mistakes and then start changing conditions (Temperature, Mg concentration, annealing temperature varying with cycles, polymerase, etc)

Also if you designed the primers based on the cDNA but the template has introns, did you check that there are no introns disturbing the sequence which the primers should be annealing to?

Answer 2

Your sequence source is cDNA so I'll ask an obvious thing to check. Do your primers overlap a splice junction? That would screw up a genomic amplification.

Depends on what you are going to do with the mutant DNA. A plasmid will have lower complexity and makes for easier PCR. Much more reliable than genomic PCR. Unless you have to clone your gene into a plasmid first. That can take some time and and have its own issues.

Answer 3

Pcr Based Mutagenesis

Answer 4

With genomic DNA the PCR should work as long as both forward and reverse primers are present. You probably need to optimise the amount of genomic DNA template present in the reaction cocktail. In case of large eukaryotic genomes the recommended amount is 100ng upwards. You could also change the annealing temperatures, try a "step-up" programme. Start with 2 cycles at a lower Tm increasing it by 2 degrees with each 2 cycle loop. The final loop with as many as 20 cycles should have the optimum Tm.

Answer 5

If you have both forward and reverse primers, the PCR should work with genomic DNA, assuming the genomic DNA has the mutation you are looking for. If the mutation is not present in the genomic DNA, your template strands will probably not anneal properly to the genomic DNA, thus no amplification.

You might try optimizing the PCR conditions if you think the reaction should work.

Answer 6

bear in mind that the polymerization continually starts from the three' end of the template! so that you temme is including a primer to 5' end of template is rather of any use? yet, yeah it will be conceivable.