I have checked the extracted DNA in horizontal gel electrophoresis and it seems oak. Freshly diluted DNA and primer before working to solve the problem but that did not help. I was getting mutilple clear bands before, but now only dimers all of a sudden. Wondering if anybody else had the same experience.
Did you change by any chance the brand or type of PCR tubes? It may sound unbelievable but I realized that because I changed the brand of the PCR tubes I started getting dimers with DNA and primers that were working perfectly before and less volume was in the tubes, probably although the thermocycler has heated lid, the new PCR tubes opened somehow. So, I came back to the previous PCR tubes and then everything started working again, another solution to try but more time consuming is to add the same volume of mineral oil in all the PCR tubes, add the mastermix prepared, briefly spin and then run the PCR program.
Good luck
I think that you have a primer dimer contamination problem. Some of your amplified material with a PD has contaminated one of your reagents and because it is small and has perfect homology with the primers it will always amplify better than the DNA template and take over the reaction. If this is the case then your zero sample control will also have a strong PD band.
you can use all new reagents and prepare the pcr in someone elses area or another lab with another researchers pipettes or you can re design one primer to fit outside of the primer positions that you are using then the PD contamination can not amplify
first of all you check positive and negative control.
in the other hand your DNA was preserve in good condition?
i think that is better u change your tube and termocyclere or work in another lab.
I have checked the dna through spectro and gel run... its ok. I have checked the chemicals with another pcr mastermix protocol using different dna template and primer...the chemicals are working fine. Checked the thermal cycler and thats ok too...Now i'm thinking suddenly the pcr mastermix protocol I've been using might have a problem.
May be problem with mastermix protocol, Mixing different reagents also matter sometimes,
simply change the primers and compare with another primer, then you will easily understand where is the problem, may be the primer problem,
Thank you all for your advices. I am happy to say after multiple trouble shooting procedures the problem has been solved. I did the following. treated DNA with RNAse and ice cold ethanol to remove inhibitory compound. Re diluted DNA as per new quantification and primer as well.. readjusted primer, DNA and taq amounts in the protocol. It seems to be working now.
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