HELP! I have a PCR amplifing problem - PCR amplifing problem (Mar/07/2006 )
[font=Arial]We work on the expression of MDR1 and MRPs (1-5) in whole blood samples from leukemia patients.
Now, We amplified all the F & R primers in the same reaction, and all PCR products were purified by running in a 1% agarose gel with TAE buffer, 80 V for 1.5 h, and here the results that we had: all the bands of the gene expression at the same length (518) !!
So what's wrong?
Is it because we use all the 6 primers in the same reaction?
Should we put them separately in every reaction for each primer?
Will U HELP me to solve this problem ????!!!
I my view point there is one reason that the difference between the PCR product is of only few base pairs so to check that try to run the product on 2% agarose gel, then see wt happens i think that they would be seprated in this way or u can also rum them on poly acralamide gel if u have that facility.
Did you contaminate your primers?
6 Primers in one reaction? can you give a bit more information. Are you amplifying with one reverse and five forwards? I can see that this is a problem as Taq will alway favour the smaller products. When you have a PCR with so many primers in it, careful optimisation of the annealing temp is required as well as amounts of reagents, i.e. alot more dNTP's are required than a standard one product PCR.
Is this a PCR you have designed yourself or a standard protocol?
What result was expected?