PCR almost like false positive problem! - How can I get rid of 5bp band near mine !!!!!!! (Jul/24/2006 )
Dear friend's I have just read this page and I found your touhgts very intresting on any issue there was a problem!!!! So i registered and I will say my problem!!!
I work on HLA B27 gene! My band is 136 bp and there is a problem from the begging ..... at the negative samples I get a band of 138bp at almost the same yeld as the positive and except from magic I can do nothing else to get rid of it!!!!! My situation is very difficult because I was asked to do this reaction for patients and I must have it ready in one month....so ..I neddd your help please!!
To make it clear .... it is not a contamination but the primers when there is no HLA B27 the primers grab to HLA B28 or an other HLA B that has 5 bp more than HLA B27 and gives this almost false positive image which is unaxceptable for the doctors and the patients and for me too.....Except from selecting new bigger primers which what are my other objectives.....
I have an option which I will try the next days....I will do the same thing as I do with gene specific RT-PCR...... which is ice water after the first denaturation (at 95 ) and the continue with thenormal pcr cycles and annealing temperature!!! Is it wrong... what do you say
I have read troubleshooting books but they all talk for longer or shorter bands, mine is only 5 bp longer and nothing works....when I lost the longer bands I lost the HLA B27 band too!!!
I have an other question for you.... Is there any one that uses DNAzol DIrect for DNA isolation from blood..... can you tell me how you do it!!!! Following the protocol the is still too many proteins in the mix that inhibits the polymarase such as heme etc..... should I boil the samples or denaturate at each cycle for more the 60 sec???
Thank you for reading it....
I think that re-designing your primers would be the best option?
Thank you for your help.... After doing my like RT-PCR reaction I used to have 5bp above mine.
So the picture now is .....
I have aslo designed new primers and the band I will get is 156 bp in stead of 136..
SO thank you again for your reply....
Goodbye from Greece!!!