Did you do gapdh/actin, etc. to confirm the extraction/PCV system is OK?

Hi,

I was about to say about primer, but you already checked. please check your new lot of reagent, especially for the ions, and add some extra cycle.

In addition, check your gel concentration, buffer. Best wishes for your experiments. I know how it feels when it shows such bands.

Are you running a no template control sample and does it give you a smear or the odd sized pcr band. Have you changed your dna isolation method .? Do you measure the purity and amount of dna and are you loading approximately the same amount of dna from all of the samples. Do you dilute your primers in water or TE ?

Thank you all for your answers!

Junjie Shao I tried another genotyping PCR of a colleague and it worked.

Shamsunnahar Mukta ok, I will have a look!

Paul Rutland yes, on both images the very last lane (separated by an empty lane) is the H2O control. As you can see on the images, on 2.9. it was clean, on 23.9. it shows two weak bands. The lower one I assumed is the primer band, and then there is a faint band a ~500 bp.

At the beginning I isolated DNA from tail biopsies, about a year ago we changed to ear biopsies - but this also worked for a while. I do the extraction using a commercially available lysis reagent and tried both, one specific for tail and one specific for ear biopsies.

I have not checked purity and amount of DNA yet. I will try.

I dilute my primers in water.

Do you have further ideas with these answers?

Although many people and companies dilute primers in water I think that it is much better to dissolve them and store in TE. Water absorbs carbon dioxide and is acidic and ,over time, will depurinate oligos and they degrade. Tris buffers alkaline so depurination does not take place and the EDTA chelates divalent ions like Mg so nucleases cannot work . I think that your primers are degrading and the inconsistency of your amplification is because there is not enough primer (full length) anymore. The smearing that you sometimes see could be because one primer is not degraded ( maybe the lower purine oligo) and you are seeing single primer extension of varying lengths.

1 Try using 4 times as much primer and see if it works as there may be enough undenatured primerleft while you re order the primers and dilute and store them in dilute TE (10mM)

I am assuming that you mix the thawed primer aliquot well before adding primer to the pcr mixture as just allowing a primer to thaw gently causes non homogeneity in the primer concentration ( water at the top and too concentrated oligo and salts at the bottom of the tube) then if you take primer from the bottom of the tube there will not be enough primer later on for future assays

Paul Rutland Thanks again for your suggestions and you time! It sounds reasonable and I will definitely go for TE-buffer insteadt of H2O in the future.

I have not found the time yet to repeat the PCR with more primer but I will give it a try this week.

Dear Paul Rutland

I repeated the PCR with new, freshly lysed biopsies using 4x amount of the primers (40 µM). See attached the result. Unfortunately, it did not bring any improvement. (The last lane is again a H2O control.)

I ordered tail biopsies from the animal facility and see if this works (and hopefully at least catch up with the genotyping).

Not only no improvement Felicia Maull but worse primer dimer and smeary amplimers. .Best I can suggest in that case is new primers again...just desalted grade are quite cheap and hope for the same improvement as last time . Primers are cheap.Animal house charges are not. Good luck