i had designed primers for two sequences using blast tools in NCBI but they gave poor amplification lower than the ladder after gradient pcr
i want any one to check them and help me in designing new primers
the sequences and designed primers are attached file
Could you give us the conditions of your PCR, like temperature used for hybridization step, enzyme used, primer and dNTP concentration, source of DNA ? Gel photo would help as well.
First sequence is really AT rich with a lot of A and T stretches, second one has a lot of C streches as well. I suggest you change enzyme, try adding DMSO or TEMAC to help the enzyme process these streches.
I would use primer3 software for primer design but from what you say your problem sounds like your primers are annealing with each other to form a primer dimer and not allowing the target sequence to amplify. Before re designing the primers try using less primer and a hot start enzyme if your lab has some which may work as it stops primer dimer from forming
Hi Noha,
I only tested the first set of primers. ok they are not perfect (see attachment I produced in Oligo7) but it's not catastrophic and you should have some amplification. I also tested your primers in the UCSC insilco-PCR tool, it gave an amplification in human WNT5A gene. if you're testing cancer samples, maybe this gene is affected or bears some variants in primers sequences.
but as Paul and Thibault advised, you should take the protocol and see what could be wrong.
fred
Hi. Dr. Noha
How to: Design PCR primers and check them for specificity
Starting with ...
ONE OR MORE PRIMER SEQUENCES
A TARGET TEMPLATE SEQUENCE OR ACCESSION NUMBER
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