PCR problem - basic PCR for plasmid amplification (Mar/16/2011 )

hey guys,

i have been running PCR for about 3 weeks but havent got my product of interest. i am working with TAT-apoptin plasmid and have designed primers (forward and reverse)for the PCR, i am also using a high fidelity PCR master mix. so my aim is to see a band around 460-500 bp (which will be my apoptin) but i keep gettin a constant band around 250bp which is absurd. i have tried different methods and even cjhanged the primers but nothing is working please help me out! time is going as this is my project and have to be finished by May latest! and as at now i am very much behind! :'((( please help me out!

thanks

Best Regards
Nina

NinaDims on Wed Mar 16 11:56:21 2011 said:

At least someone in as difficult situation, as I am.....

I am the newest here (few weeks old), but if I had this problem.....I would think first about contamination and problem with amplification.
Do you have this 250 bp product also in your negative samples?
When I test my new primers, first I load only negative samples (with water, no DNA) with both primers (R and F), only R and only F. This tells me if the reagents are clean of contamination and if the primers cross-react.

Did you try the BLAST search for primer specificity?

May be this link will help:
http://www.med.yale.edu/genetics/ward/tavi/Trblesht.html

Good luck! with hope to see what the more experienced will say.

Hi Nina, it will be helpful if you stated how you do your PCR: the thermal cycling conditions, the mastermix compositions, the optimization you had done etc. Maybe a gel will do as well.

To begin with, Is there any unspecific binding?