Template Amount for PCR Amplification - (May/11/2011 )

Dear All

I am trying to amplify my insert (a full length gene) from a plasmid. However, When I tried with around 3ug template, it didnt got amplified. I also checked lesser amounts, but All I got was the primer dimers.

What is the optimal template concentration I can use?

Thanks for your help

-dreamer0085-

3 ug is a massive amount. You should only need between 0.1 and 500 ng.

-bob1-

Thanks

I tried 0.1 ug too. I made a master mix and then aliquoted into several tubes. It should be enough ?! But still I got only primer dimers!

-dreamer0085-

what is the buffer in which you have/dilute the template? if it is te then you may be inhibiting the pcr reaction by chelating too much mg.

are you sure the primers are correct for your plasmid or insert?

-mdfenko-

No I diluted my template (Plasmid) in water. I am fairly sure about the primers. I tried with a different set of primers. and didnt get anything

-dreamer0085-

0.1 to 1 ng is good amount for a 50 ul reaction. Experiment different amount of template. Best source is to check instructions for that particular polymerase, comes with each enzyme. you may want to increase initial denaturation temp and time, or cut your plasmid. it's more difficult to melt supercoiled DNA. of course, do not forget a linear positive control that you are preparing with a master mix. good luck!
http://www.bioprotocols.info/reagent_and_buffer_recipes/index.php

-chromatin-

dreamer0085 on Wed May 11 23:22:51 2011 said:

3 ug is a huge amount for a plasmid. It corrsponds to millions of copies depending on the size. use no more than 1 ng for plasmids.

-Bassaml7-