Why is 3 mg Too Much for PCR Reaction?

[karthik3k]

I used 3mg intsead of 10ng template for PCR reaction.

And i didnt find any product when i ran it on a gel.

Can anybody explain why this happens ?

Plz don't say its due to impurities...
Iam quiet sure abt the sample i used ...

THanx

 

[Monique]

You mean 3 ng? You are on the low end, but it still should be enough. First questions: did the PCR work before, did the sample work before, did you include positive controls in the protocol?

 

[karthik3k]

Yes.
I used 3 micro gram

 

[iansmith]

Too much DNA template is inhibitory due to competition for annealing of primers and dNTPs. So you end up with a lot of short fragment because dNTPs is depleted quickly. It should also be noted that the probability of nonspecific primer binding is increased due to change of the optimal quality to a suboptimal quality.

Also large volume of template migth carry "garbage" from the DNA isolation step, depending on the methode use and you. This "garbage" migth inhibit the enzyme.

 

[Monique]

I used 3 micro gram

Ah, you can tell from my asumption it must've been 3 ng.. that 3 mg really is way too much the normal range would be 10 ng - 500 ng, less is better since you get less contaminants and the reaction overall is more efficient.

Ian said it well, your primers are binding all over the genome since there is so much DNA to bind to, your dNTPs get depleted, and you are introducing contaminants by adding such a large volume (remember DNA binds proteins).