I'm currently conducting PCR on a gene and since my DNA is bisulfite treated, I have spent a considerable amount of time standardizing the PCR for the gene. I have been consistently getting good results in the PCR when I use a small number of samples e.g. 5. But now, when I've increased the sample number the PCR amplification is nil. I'm getting a clean PCR with only the ladder visible.
What could be an underlying reason? The mix, machines, samples are all the same and I've gotten results in them.
Did your positive control work? That would rule out primer degradation/machine issues/etc. Let us know if you have been able to rule out any of the variables.
Try to change one component at a time and see where is the difference. You might get result. If you have already tried that one, then check
1. added DNA or not (sometimes it happen)
2. centrifuge your mastermix and DNA
3. prepare working primers again from stock
4. if sharing primers with others, then order new set of primers
5. use DNA (which gave result in the previous PCR) as control in the current PCR
6. check loading dye concentration
7. pH of gel running buffer
8. TaqPol - sometimes, it kept at room temperature for long time (denatured)
1) I would check the DNA quality and quantity
2) Try to change one component at a time in PCR say dNTPs old vs new, primers old-vs new etc.
DNA quantity might be the source the problem. Minimum of 10ng usually works best
Out of all the ingredients in the pcr reaction, the one that tends to cause no-bands (and brings back bands when replaced) most often in my hands has been dNTPs. They are the most thermo labile reagent, so if you have taken the same aliquot out of the freezer multiple times there is a high chance they simply expired due to heat/light exposure.
Beyond this, my next suggestion would be to double-verify that the rxn is getting a viable template (correctly scaled ng amount of gDNA per rxn).
dNTPs are indeed the most labile component and you should try using a fresh aliquot. Next thing that you need to try is primers.
If the primers are dissolved in water then they may have depurinated in the slightly acidic solution and degraded. Try fresh primer from stock or order another batch
First, I would have checked the quality of the DNA using nanodrop. The second possible contributing factor could be the temperature sensitive deoxynucleotide solution, which should be stored at -20 C and have minimal exposure to ambient temperature. You can make sure that your dNTP is functional using a control DNA sample. Finally, you should control the pH of your gel running buffer.
Did your positive control work? That would rule out primer degradation/machine issues/etc. Let us know if you have been able to rule out any of the variables.
Go back to doing the small sample number again, to see if it works, if it does then you are forgetting a step in your preparation but if it doesn't then you have to troubleshoot.
I am not sure about the DNA content.A nanodrop may help to answer this question. If ensures the presence of DNA than ur primer may be relooked for its validity
Quality of the templale DNA in terms of both concentration and purity is an important issue. Sometimes using old aliquots of dNTPs and primers may be responsible, particularly if freezed and thawed for several times.
Check your RNA integrity on gel electrophoresis, it may be that your RNA become denautred.
Use positive control in your experiment it may help, Use additional 0.5ul of DNA Taq polymerase at the end of your PCR mix.
Getting results for small sample size means your primers, machine etc are working, but preparing large samples for PCR may expose heat sensitive reagents to ambient temperature for long time which may effect the reaction.
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