I have a PCR problem and I hope to get some ideas here to resolve them. I have been looking around for solutions but as every PCR rx is different i'm still troubled.
I'm a masters student trying to sequence the TP53 gene with 10 exons and 7 primer pairs. (primers: 2-3, 4, 5-6, 7, 8-9, 10, 11). I have a total of 40 samples of genomic DNA from cancer patients, normal and tumor tissue.
The protocol i'm using is called TP53SequencingProtocol_ABI3730_DNR.doc
DNA 25ng/ul 1 ul
Primer 5’ 6uM 0,5µl
Primer 3’ 6uM 0,5µl
dNTP 2,5mM 0,75µl
PCRbuffer with MgCl2 1,0 µl
HotStar Taq 0,1µl
After running PCR on all 40 tumor samples in all 7 primers I have gotten some consistency in bands for most of the samples, while some samples never show any bands. The primers i get bands in are 2-3, 5-6, 7 and 11.
Primer 4, 8-9, and 10 has never given and bands for any sample.
So yesterday i made new concentrations for primer 4, 8-9, and 10 at 4µM and 8 µM (I have been using 6µM) and tested with only one tumor sample (because i ended up making too little PCR mix for all my intended test samples) that had the most intensity in the 3 primers that showed bands. This sample has not given any bands for either of the 3 primers 4, 8-9, and 10 at 6µM . But for my test at 4µM and 8µM this sample showed bands for all the primers 4, 8-9, and 10.
So today i tried all my samples with primer 4 at 8µM concentrations and this time i get no bands again.
However i do get some smear of primers, and DNA, and a darker border of sybrsafe migrating instead of binding to DNA. I have also been using the same PCR machine for all runs.
So it is evident my PCR mix is acting up. I have another PCR of primer 8-9 at 8µM waiting to run gel tomorrow . After that i'm trying more samples with primer cons. 8µM as one test sample is ridiculous
, although my intention was just to check if i get bands, which i did yesterday.
So, i will be grateful for any help on advice and I will post update when i figure it out.