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PCR-tp53 exons

PCR-tp53 exons - PCR - Polymerase Chain Reaction Forum

PCR-tp53 exons - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 04-27-2011, 05:11 PM
Pipette Filler
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Default PCR-tp53 exons



Hi,

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

Reagents volume/
quantity
DNA 25ng/ul 1 ul
Primer 5 6uM 0,5l
Primer 3 6uM 0,5l
dNTP 2,5mM 0,75l
PCRbuffer with MgCl2 1,0 l
HotStar Taq 0,1l

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 4M and 8 M (I have been using 6M) 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 6M . But for my test at 4M and 8M this sample showed bands for all the primers 4, 8-9, and 10.

So today i tried all my samples with primer 4 at 8M 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 8M waiting to run gel tomorrow . After that i'm trying more samples with primer cons. 8M 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.
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