I have designed primers using the mRNA sequences provided by ncbi with Primer3 and primer blast from ncbi for my target genes. I have checked those primers with in silico PCR with USCS and they have found my target gene. Then I have designed gene fragments which are supposed to be recognized by my primers and getting amplified. I have checked these gene fragments against my primers. I performed an Eva Green qPCR experiment testing 10, 50 and 500 copies/µl of the gene fragments and did not receive any results. Where is the mistake?
The PCR-protocol was a standard protocol which are used in our lab:
2 min 95°C
45 cycles
30 s 95°C
20 s 55°C
45 s 60°C
Thank you all for your help.
Nicole
Dear Nicole
Not the answer, but just some thoughts:
1) Did you include any sort of control to test if you polymerase works properly under your conditions?
2) How long are you gene fragments?
3) Did you check your primers for self-complementary?
1) no
2) btw. 200 and 400
3) yes, they are not self-complementary
I think we already found the problem. The gene fragments were probably too much diluted.
What are the optimal reaction conditions for the polymerase you used in qPCR? A 60*C extension step is rather cold, normal is 72*C. Also, many qPCR mixes have a stabilized polymerase that needs 5-10 min at 95*C as a first heat step. Check the instructions for the kit and see if you need to adjust any of the cycling steps.
Good luck!
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