Assuming that your experiments with Taq and Q5 are repeatable, the only thing that remains would be the magnesium ions. The magnesium ions form a gradient in solution when freezed, and need to be throughly mixed before use.

Contamination is also a common problem. Make sure your tubes and water are nuclease-free.

I agree with Danial Yousefi about thawing Mg and this can be a bad problem if you remove selectively the high concentration of Mg at the bottom of the tube a few times then even mixing may eventually fail as too much Mg has been removed.

Another possibility is the template dna. If you have changed anything in your purification method then you may have pcr inhibitors in your dna. Measure the od260/280 and also try adding more MG and see if this improves the amplification. Yes I do think that the primers are annealing at too low a temperature. Consider BLAST ing the primers and designing longer primers by adding appropriate bases at the 5'end of the primers to get an annealing temperature of nearer to 60c

Hi Gen Gi

Have you tried changing the Taq PCR buffer? If you have not, then try a PCR reaction with new buffer. I was having this problem recently, i changed the buffer and it started working for me again. I hope it help you.

Thank you very much for the replies. I was very, very careful to mix the new taq reagents before use so I don't think that's it. I agree, mixing is very important and not mixing has caused many problems in the past. I didn't know it was because of Mg though -- that is good to know.

I looked at my recipe for Q5 and taq. The main difference was that I use higher concentration of primers for Q5 (I don't know why, just the protocol I have).

I repeated the PCR with taq and the non-working primers with 2 conditions:

1. With my normal concentration of primers for taq (0.2 uM final): No band

2. With 5X concentration of primers (1 uM final): Good band

So my primers are the problem? Why do I need higher concentration of primers now for bands? The old concentration (0.2 uM) used to work fine. The primers were stored at -20 at 100 uM and I didn't freeze/thaw them more than a handful of times.

I've heard polymerases can sometimes degrade primers, but NEB says taq will not degrade primers.

I purchased brand new primer stocks and this did not fix the issue of no bands.

So previously 0.2 uM primers was sufficient for good bands.

Now 0.2 uM is not enough and I only get bands if I use 1 uM primers.

Why is this? NEB says primers for Taq can go as low as 0.05 uM...

The Taq/buffer are brand new...

The primers are brand new...

The polymerase recipe I follow is exactly the same...

The thermocycler program is exactly the same...

It happens if you dilute and store your primers in water not TE.Water absorbs CO2 from the air and becomes acidic.The single stranded primer depurinates and is degraded. TE buffers at alkaline pH and the edta removes divalent ions so stops nucleases from working. If you needed to add more primer then it suggests that most of your primer is degraded

But this repeated with absolutely brand new primer stocks.

Even with brand new primers directly from IDT, I still need 1 uM, not 0.2 uM.

Even though 0.2 uM used to work fine.

Is your pcr on single species genome or are you looking for many organisms many of which amplify?

Are your primers single sequence or do they have many degenerate bases to amplify more than one species? Do they contain Inosine in place of Ns in the sequence?

Does the paperwork that IDT provide show that the base addition of the oligo manufacture is greater than 99% at each bace?

Check your primer concentrations with either a nanodrop or a qubit flourometer or your spectrophotometer

I use the primers for a couple species within a specific genera, but pretty much all my PCRs have been with a specific lab stain of one species (the old and new PCRs were done from the same plate of bacteria). The primers have no degenerate bases, nor any inosine -- the primers are solely unmodified ATGC. Is your idea that the frequency of specific bases can change between primer orders? I think that's a very interesting idea and I thank you for informing me of that issue.

I haven't been able to find the documentation about base frequencies unfortunately.

I nanodropped my primers. I follow IDT's given instructions for resuspending at 100 uM. After nanodropping:

Old primer stocks (that used to work at low uM but now only work at high uM): ~60 uM (instead of the intended 100 uM)

Newly resuspended primer stocks (that only work at high uM, not low uM): ~60 uM (instead of the intended 100 uM)

I find it odd my primers were not at 100 uM, however, the fact that both new and old stocks had the same values leads to me conclude it's probably not the sole issue.

After completely replacing:

1. Taq+buffers

2. Primer stocks

3. Water

And checking the thermocycler+PCR recipe for the 10th time, I've concluded it must be my DNA. Unfortunately, I do not have any old DNA that used to work with 0.2 uM primers.

I have tried many, many different DNA preps this week and all fail with 0.2 uM primers. Considering some had quite high levels of DNA, I concluded it is likely there is some inhibitor in my preps.

I diluted one DNA prep: 1:10, 1:100, and 1:1000. The 1:10 gave a faint band (with 0.2 uM primers), while undiluted DNA and greater dilutions gave no bands.

Therefore, I conclude my DNA preparation process has become dirtier, although this can be overcome by adding more primers.

I am surprised by this as some of the DNA preps that failed came from bacteria cultures that were very low density. (For the PCRs, I did not add add greater volumes of DNA for the low DNA preps).

I don't know how my DNA prep has changed. My current idea is that I've started vortexing the bacteria much harder than I used to before harvesting their DNA... and that is releasing inhibitors I suppose?

It was not the frequencies of the bases but the efficiency of incorporation which is measured in production by the amount of trityl produced at each base addition, If low then you have lots of fail sequences and if >100% then you have branched oligos and both of these situations will cause poor binding and amplification, Tech support at your oligo maker will be able to see this but these are uncommon these days,

The inosine thing was that some high fidelity polymerases do not work well with inosine

The mixed base was that if there are many degenerate bases there are a lot of different oligos not just one and this can cause problems

Do another check that the primer sequences have not changed.

It seemed possible that a base mismatch at or near the 3;end of the primer would cause pcr failure and I wondered if other species had become common and were interfering with pcr.

Ok that’s just thoughts, It sounds as if you are right that you have pcr inhibitors. Most of these extract magnesium from the pcr mix. I would double the amount of magnesium that you normally use and see if it works. Asa test for inhibitors take a dna that does amplify and add poor quality dna to it. Amplify the 2 dna samples and if adding dna to the working sample causes it to fail then you have inhibitors. Sometimes adding BSA works and sometimes just diluting the dna until it works is a good try as you have done. Good try.

Concrning dna prep do you need to do a dna prep or can you just pick a colony,add it to the pcr mic and amplify?

you may find the attached paper interesting

False negatives are extremely deleterious to my project, so I will need to optimize the conditions to avoid this in the future.

The DNA prep involves little more than boiling bacterial culture/colonies and adding that to the pcr mix -- ie, basically colony PCR.

Is it still likely the inhibitors are Mg chelators? If so, I will titrate in MgCl2 to find the most robust formulation for amplification from dense to dilute samples.

I will read the attached paper now.

It is likely to be inhibitors as your dna dilution experiment shows. Mg titration is a

very good idea and if this fails then consider less dna and more cycles . There are enzymes (KOD polymerase) that work well in dirty mixtures but this should not be necessary as the assay has worked prviously

good luck

yes dear