I've ran a PCR using Hot start Taq polymerase instead of normal taq polymerase by mistake, but as the result i have found some pure and non-sharp bands, may any one help me to find why it has happen, plaese?
thanks.
note: DNA concentration was pure. but when i run it by normal taq, I have no problem at all.
In colony (mycobacteria, nocardia spp.) PCR, the optimal temperature for Hot stat is largely based the kit manufacturers recommendation. But usually about 95 degrees Celsius for 10min depending on the enzyme stability.
Hi Mr. Aref
In hot-start PCR, Taq polymerase is inactive until heated. Hot-start PCR activation approaches allow users to minimize non-specific amplification while increasing target yield and specificity.
Did you use Read-mix enzyme or just the DNA polymerase and the rest (dntp, mgcl2 etc) were added manually? Since they activated through high-temp during pre-denaturation, some of the reagents are kept separate until the mixture is heated to the specific annealing temperature, if you added the dntp, buffer manually. But if used the ready-mix, some of them can be mixed directly from the start.
I have been using hot start enzyme for 4 years (Bioline, UK) and it always better than conventional taq. No special PCR condition except the pre-denaturation time (for activate the enzyme). The activation time (predenaturation) is about 95 degrees for 2-3 minutes before the cycling step. The annealing time is about 15-20 sec, and 72 degress for extension 20 s - 1 min (depends on the amplicon size).
May this answer help
Thank you
Hi,
Difference between Hotstart Taq and normal taq is that hotstart taq will be active only at higher temparature (Generally Incubated at 95deg for 5-10mins for activation).
We can expect better results with hot start taq as the taq is inactive at the PCR reaction set up temparature, this will prevent primer dimer formation.
https://youtu.be/tNR7Curs0N8
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