The query is that when I initially performed RT-PCR with my specific primer at a given set of conditions, good results were obtained (i.e no primer dimer formation observed when checked through agarose gel). But as the same experiment was repeated again and again (on daily basis), primer dimer formation was there. Following are the steps I have tried for troubleshooting without any positive outcome:
Step 1:
Tried using different additives like BSA, glucose, tris-EDTA.
Step 2:
Also tried increasing annealing temperature by 1 degree C but no effect.
Step 3:
Fresh but same Primers were tried (from IDT), but still resulted in Primer-Dimer formation.
Here are the conditions I am using in my experiments:
Sample - Plant roots stored at -80 C.
RNA extraction using Qiagen Kit
cDNA synthesis using iScript
RT-PCR annealing temperature was - 60 C
There are answers within research gate:
https://www.researchgate.net/post/How_to_avoid_Primer-Dimer_Formation_and_get_our_gene_amplified
Best,
S
Get your primers purified by PAGE
also don't store primers in H2O, use TE
If you've been using the same thermal cycler for all your experiments and the primer dimer formation has slowly gotten worse, then your problem may be that the thermal cycler itself is not operating properly. Try a different one and see what happens.
My experience with additives is that DMSO can be a reaction killer and that 1 M betaine is much more effective. (Make from betaine anhydrous.)
And since primers are cheap, ordering some alternative pairs and testing them may be the simplest, fastest solution.
The best annealing temperature could be determined using a gradient pcr. Here you can vary the annealing temperatures in different reaction tubes.
Your primers may also be the problem. It might be best to design new ones and avoid primer formation. Here is a webtool that could help predict the chances of primer formation.
https://www.idtdna.com/calc/analyzer
Also your primer concentration and volume in the reaction mix might be too high. Try reducing them. A chessboard PCR is a good approach to vary primer volume and magnesium concentration.
Finally try a different thermocycler and compare results.
I hope this helps
A gradient PCR with a range of 10-12degrees Celsius temp. variance would be worth trying if your primers are prone to cross reactivity, a single degree temp change might not be enough.
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