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[Stewartsj]

Hi all,

I've been contemplating on whether or not I should reach out to the internet community for help on my PCR problems; it seems like a last resort having to seek out other people's help but still haven't been able to solve anything.

I've been trying to just investigate whether or not I have certain calcium channels in my vessels. I first went to design real time primers to perform real time PCR. 3 sets of primers later, my results were: some samples showed expression; while some did not. Control tissues however were alright. My samples that I was testing however was very inconsistent. We then tried to increase the number of mRNA available by pooling more vessels, making sure all the extraction techniques were done as soon as possible, and increasing the amplification cycles. Same results; sometimes none of my wells showed up.

I'm now moving on to constructing nested primers to perform RT-PCR, as real time PCR was really getting expensive. I have constructed successfully inner primers, but I"m having trouble with outer primers. I've done the following:

1. Construct inner primers, identify the sizes and where in the sequence is being amplified

2. Using primer 3, include a parameter that specifies the primer being made includes the sequence that the inner primer has amplified

3. Double checked on NCBI primer blast to see if there are any potential unintended products. Chose one that had the least amount of unintended products.

- After 2 sets of primers, I'm still having no luck. What should I do? I'm currently using Taq polymerase, using 0.1 µM of primers, 12.5 µL PCR Master Mix in a 25 µL tube per reaction. I've double checked over and over on both primer 3 and ncbi primer blasts, and testing them on control tissues; and yet I still do not get any expressions.