I'm doing 16s rRNA amplification using DNA extracted via the boiling method for several bacterial samples.
The quantities of each PCR reaction are;
DNA template - 1 ul
Forward primer - 1 ul
Reverse primer - 1 ul
Nuclease free water - 9.5 ul
Go-Taq Green master mix - 12.5 ul
5 ul of each PCR reaction is loaded into the 1.5% agarose gel.
Clear bands can be observed for 7 of the 9 samples, as well as the positive control. However no band is present for one, while another one is present but fainter than the rest.
What could be causing this?
Some possible reasons for no band visible relate to there being no pcr product to see. This can happen with
1 too little dna in your sample.
2 poor quality dna with pcr inhibitors present in the dna that failed to amplify.
3 The failed dna is good quality but there is a polymorphism at the 3'end of one primer so one primer does not anneal so no amplification.
Similarly the faint sample can also be that there is too little dna or too much dna with pcr inhibitors present but in smaller amounts so there is some inhibition of the pcr reaction.
You can test for inhibitors by mixing a sample that works with one that does not work and if the pcr fails then the failing sample probably has pcr inhibitors. Alternatively try diluting your failed dna in water 1:2 ,1:4 and 1:8 and if the pcr works at higher dilution then the dna is probably dirty and contains inhibitors. Another possibility for dealing with dirty dna samples is to add mor magnesium as many inhibitors remove Mg from solution or to use a polymerase that tolerates dirty dna better
test a separate control sample that simultaneously tests the materials you are using If the substances required by the enzyme are not sufficient, the reaction will not take place to a noticeable effect. Use good enzymes and ingredients I use cheap enzymes and masters belonging to Kiagene Fanavar Company 1ml=4 USD, which I have seen very acceptable results. I will send you the link of these materials, maybe it will be helpful.
https://kiagene.ir/store/product/2x-taq-master-mix/
The fact that your positive control was successful means that the reagents, thermocycler program, etc are all correct & able to amplify DNA.
That means that the failure to amplify is unique to that DNA sample. I'd recommend trying a few different concentrations of that DNA (sometimes less is actually better).
Good luck!
Add one positive control and one negative control with your samples during PCR
Please check your primer. prepare another primer. I had the same problem.
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