I am working of T. thermophilus bacteria and i want to amplify SufS gene, but always i just got the dimmer at the end. i change the primers but same problem, i just got dimmer, any one please?
Hi Abdul,
Jeffrey is right all companies that sell taq will sell a hot start taq and one of your colleagues will have some to test. If not you can do a hot start with any taq by making your pcr mixture with everything except the taq and heat the tubes to 70 c or more. then add a small volume 1-2ul of the taq into the tube with a fresh tip each time and start cycling. This way the mix never gets below about 55c when the enzyme is present so no primer dimer forms. If this works then probably a hot start enzyme is a good idea for your reaction. best wishes, Paul
4 types primer i used for it so for. annealing temp. 55, 53 and 50 each individual test. denaturating temp. 95C and extinction temp. 72C. the cycles are 30x and 35x respectivily.
use a hot start enzyme then the primers are never together with an active enzyme at the low temperature needed to produce dimerisation
@Abdul Majid
There are many hot start enzymes from which to choose. Enzymatics had their Phoenix Taq, Bioline has Immolase along with a couple others, ThermoFisher has several including Platinum Taq, NEB has 2 or 3 including OneTaq. You might find a colleague in your building or complex that already has a hot start enzyme so you might not need to order. In fact, I suggest if your colleagues have to share that you test several as not all will perform the same. Good luck.
Hi Abdul,
Jeffrey is right all companies that sell taq will sell a hot start taq and one of your colleagues will have some to test. If not you can do a hot start with any taq by making your pcr mixture with everything except the taq and heat the tubes to 70 c or more. then add a small volume 1-2ul of the taq into the tube with a fresh tip each time and start cycling. This way the mix never gets below about 55c when the enzyme is present so no primer dimer forms. If this works then probably a hot start enzyme is a good idea for your reaction. best wishes, Paul
@Qing Zhao .. i have used Pfu enzyme mix.. yes i used gDNA extract from HB8 bacteria.
i used this enzyme with instruction protocol as well as change its protocol but didn't get the results, than used anther enzyme taq but the result is same. now i try KOD hot enzyme today see what happened ?
Thanks a lot of your help. today i get target bar on the specific location. the problem is in enzyme i use Kod hot start enzyme, other stuff is the same. Thank you very much @Paul Rutland @Jeffrey Shelton @Qing Zhao
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