I am doing genotyping to identify mutants. i use HD2B gene primer which are not working very efficiently. they work some times for some of the samples to barely amplify the gene (see figure attached) . I have ordered these primers about couple months ago and these primers i got from one of the publication, i have tried pcr with almost everything new, like new reagents with new primer dilutions,. DNA samples are even fine, they are pretty ok with other gene primers as you can see in the figure. Fig A1, u can see band in +ve with HD2B primers, but in most of the samples. In fig B1, band are very faint and almost not noticeable. best solution can be ordering new primer after making sure their specifictiy and other related things. but i am just curious why this is happening so. lower figure are with same DNA template and same regents.
The small bright band at the bottom of the gel is primer dimer where the 2 primers anneal together to form a short product. This dimer melts easily and amplifies well so takes over the reaction and eventually removes all of the primers from the reaction mix so the proper product cannot form.. There are remedies. 1 use much less primer. Try a reaction with the same dna but dilutions 1/2, 1/4, 1/8 and 1/16 amount of primer in each tube. I expect the reaction will work better at one of these dilutions
If you can find a hot start enzyme then primer dimer will not form and the pcr will work.
If not then try a manual hot start where you make a sample and reagent mix with all reagents except the enzyme. Bring this to 80c and hold this temperature. Add the enzyme ( diluted for pipetting) to each tube and immediately cap the tube and start cycling.
Hot start will work but if it does not then use primer3 or similar software to design good primers that do not form primer dimers
Please note that the primer dimer is not hugely bright but it is very short so does not trap much ethidium bromide so in reality there is a huge amount of dimer in your samples
The small bright band at the bottom of the gel is primer dimer where the 2 primers anneal together to form a short product. This dimer melts easily and amplifies well so takes over the reaction and eventually removes all of the primers from the reaction mix so the proper product cannot form.. There are remedies. 1 use much less primer. Try a reaction with the same dna but dilutions 1/2, 1/4, 1/8 and 1/16 amount of primer in each tube. I expect the reaction will work better at one of these dilutions
If you can find a hot start enzyme then primer dimer will not form and the pcr will work.
If not then try a manual hot start where you make a sample and reagent mix with all reagents except the enzyme. Bring this to 80c and hold this temperature. Add the enzyme ( diluted for pipetting) to each tube and immediately cap the tube and start cycling.
Hot start will work but if it does not then use primer3 or similar software to design good primers that do not form primer dimers
Please note that the primer dimer is not hugely bright but it is very short so does not trap much ethidium bromide so in reality there is a huge amount of dimer in your samples
thanks Paul Rutland for taking time to detailed answer. So, you mean making further dilutions from the working dilution (1/10 from stock) which is usually used for the pcr. And these primers for genotping have been reported in a publication for this gene.
Yes you are using too much primer so further dilutions starting from your current amounts.. They may be published but the original authors may either be using a hot start enzyme or simply have not designed very good primers. I expect that using less primer will work and it is just a matter of finding the right amount and depending on your stock concentration you may have to dilute by up to 1/100
I totally agree with both of the answers of Dr. Paul Rutland. Try to do as he kindly suggested you and you'll get better results.
Best of luck
In addition to Dr. Paul’s suggestions, please also check the correct melting temperature (Tm) of the primer. I usually fix this kind of primer problem by setting the appropriate Tm to amplify the correct signal I need using Gradient on PCR. I hope it works!
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