If the second sample set is different from the first set then there is a possibility that 6 of the 8 samples have a polymorphism under one of the primers so the primer binds badly.

If you repeated the same samples then some possibilities are degradation of primers if they were made up in water not TE so only the most pure and correct concentration of dna is amplifying.

You may be annealing at too close to the maximum annealing temperature and small variations in volume of reaction mix and amount of dna are too close to the edge.

The peltier block of the pcr machine may be failing so that some samples have sub optimum cycling conditions.

Your second set of samples may have pcr inhibitors which remove Mg ions making the Taq inactive

You may have a problem making up a home made pcr mix. Make sure that you thaw all reagents like MG completely and mix them before pipetting. If the Mg is partly thawed then you have 2x MG in solution and a water iceburg floating in the reagent.Then pipetting removes 2x Mg and if done a lot means that the Mg becomes too dilute.

I would thaw a new sample of primer and also use MG from another worker. Make up a mastermix with 50% more Mg ,check that nobody has changed your pcr cycle and run a new pcr with an annealing temperature 2c lower than you now use.

Take one of your failed samples and measure its OD260/280 and run dilutions of this dna at 1:2, 1:4 and 1:8 dilutions in case you are running too much dna which is inhibiting the pcr reaction

Greetings!

This is something that can vary depending on several factors.

1- It is worth repeating the test and checking if there was any error in pipetting or mixing reagents during the PCR reaction. In fact, in general, molecular biology results should be confirmed at least 2-3 times to ensure that they are true and reproducible.

2- PCR reactions are extremely sensitive, so if you are using small amounts of DNA, the ideal is to check if the DNA was correctly added to the samples that had been positive and became negative.

3- I think it is unlikely that there was degradation of the reagents (buffer, MgCl2), but other things are easier to degrade, such as TaqPolymerase, dNTP and DNA samples. Since you continued to get positive results in the second experiment, if something degraded, it was probably the DNA samples. In some cases, depending on the type of DNA used, it is worth performing the PCR reactions in a climate-controlled room (18°C) and on ice, keeping the minimum of sensitive samples (TaqPolymerase, dNTP and DNA samples) out of the refrigerator.

I hope I have helped and I wish you luck with your experiments!

Best wishes,

Cassio!

As you got amplification in positive control and one sample, the most likely cause for no amplification in the remaining samples (that showed amplification previously) is poor quality of template DNA (Either due to using a crude DNA extraction method or due to less carefull purification that may lead to presence of contaminants in the extracted DNA like phenols that hinders PCR reactions. Or the contaminants like nucleases could degrade DNA overtime). Repeat the PCR with freshly extracted DNA. Hopefully, it will work

several factors could be responsible for this change, like degradation of sample,

viability in the sample,

gel assay system may be bad or detection method sensitivity altogether.

Contamination maybe the reason. Asthe test is very sensitive, even a small factor may cause the error. So repeat the experiment and recheck the results. Also use freshly prepared chemicals for the work