Pendragon04

I had the same experience a while back. I knew that my primers were specific for my gene of interest but when I sent the products off for sequencing, I got totally different hits. If you're not getting the right amplicon size when you conduct PCR, then chances are you're not getting your gene of interest. Possibly because the DNA template you're using might have some mutations in that gene or the primers are not that specific. Or like what the others said, you need to further optimize your conditions. Also, another advice is you need to open the sequence chromatogram file and check whether or not the automatic base calling was correct. If your PCR products weren't purified or if it's too small, the polymerase from the sequencing reaction might not be able to compliment the correct base or there could be some degradation. Anomalies like this often happen. So for endpoint PCR, ideally you'd have to send products more than a hundred base pairs long to account for end degradation. Also, make sure that the ones you send do not contain nonspecific products as this could interfere with your results. You can do gel extraction or there are spin columns available to separate the PCR mix from the products and deactivate the polymerase and other enzymes you used in amplification.

hi. do you know if platelets respond to mtt assay? what other techniques or experiments can i use to determine viability of platelets? will platelets last 3 days in a culture plate given standard media and conditions? also, how do you count platelets? can i use a hemacytometer? thank you!