Dear researchers,
I am trying to do molecular sequence of the members of Kumanoa sp. (Freshwater red algae - Rhodophyceae). And i am using the primers F160 and rbcLR (which is used by Stewart et Vis, 2007). And i am using pcr setup denaturation 95℃- 1.30mins and 35 cycles of 93℃for 1 min, 50℃ for 30 sec and 72℃ for 1 m. With final extention of 72℃ for 10 mins.. i have changed the annealing temperature of 49℃ and 51℃ but not succeeded.
Kindly suggest me ideas options for my successful dna amplification..
Where i can make changes to get a successful dna amplification...
Hi Elaya,
You can run the gradient PCR to optimise annealing temperature. Also check with the MgCl2 concentration.
Dear Laurence sir,
Thank you so much for your detailed answer. Since i new to this technique i will take some tym to understand some of your points. But i hope surerly i will do successful amplification soon..now i am getting more confident..
Thank you sir
Elaya
Dear Laurence sir,
Thank you so much sir. All your links too have good informations. I will try tounderstand the issue sir. Since i dont have gradient pcr i will run seperate pcr programe for each temperature sir. i will check my CTAB method also for some modification.as you mentioned some inhibitory molecules may present in my dna product. Other informations also i will use sir..
Thank you sir for giving me a detailed answer sir..
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