PCR, qPCR, and RT-qPCR

Top Ten Tips for Successful PCR

Posted on by Kelly Grooms

We decided to revisit a popular blog from our Promega Connections past for those of you in the amplification world. Enjoy:

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    • Modify reaction buffer composition to adjust pH and salt concentration.
    • Titrate the amount of DNA polymerase.
    • Add PCR enhancers such as BSA, betaine, DMSO, nonionic detergents, formamide or (NH4)2SO4.
    • Switch to hot-start PCR.
    • Optimize cycle number and cycling parameters, including denaturation and extension times.
    • Choose PCR primer sequences wisely.
    • Determine optimal DNA template quantity.
    • Clean up your DNA template to remove PCR inhibitors.
    • Determine the optimal annealing temperature of your PCR primer pair.

[Drum roll please]…and the  most important thing you can do to improve your PCR results is:

  • Titrate the magnesium concentration.

Choosing the Right Reverse Transcriptase for Your Project

Posted on by admin

There are a lot of choices when it comes to reverse transcriptases.  Choosing the correct one for your cDNA synthesis and RT-PCR project is important.    Here are a few questions that will lead you to right RT for your application: Continue reading “Choosing the Right Reverse Transcriptase for Your Project”

Top Ten Things You Can Do to Improve Your PCR Results

Posted on by Kelly Grooms

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10. Modify reaction buffer composition to adjust pH and salt concentration.
Continue reading “Top Ten Things You Can Do to Improve Your PCR Results”