Given that your positive control is working, that means the issue is with the tail DNA samples.

I know it sounds counter-intuitive, but try diluting the DNA 1:10 and 1:100. If you used a "quick & dirty" DNA extraction method (no column), then you might have PCR-inhibiting compounds. Diluting the DNA in water can remove enough of those to allow the PCR to work.

Good luck!

I agree with Katie A S Burnette about the pcr reagents and about pcr inhibitors especially about using less dna.

One other possibility is the quality of the reagents used to lyse the tail sample and prepare the dna. For instance if using a hot alkali method then NaOH and KOH both absorb CO2 from the air and become carbonates which will be bad for the dna preparation. It may be worth preparing new dna isolation reagents and using these in the dna preparation

I agree with the above answers.

To overcome the PCR inhibitors present in the query DNA samples, you can try modifying your DNA extraction method, you can go for a column-based DNA extraction method, and your existing DNA samples can be purified using magnetic beads / nucleic acid purification kits / column-based purification methods.

You can also check the DNA concentration of your query samples prior to setting up the PCR to see if enough DNA template is going in the PCR reaction mix.

All the best.

It's probably a DNA issue. The primers and PCR conditions and gel electrophoresis all work so all those parameters are fine.

If there was any alcohol carryover from your DNA prep then the PCR will be inhibited. Out of curiosity, do any of the samples exit upwards from the well when loaded? That's a sign of high levels of carryover of alcohol.

I'd suggest getting new DNA extraction reagents, try the extraction again, quantify the DNA, and try a few different dilutions of DNA for the PCR.

good luck!

Hello Maria,

several years ago I helped a colleague by genotyping their mice because she had the same problem.

First: Genomic DNA around 50 ng/PCR reaction

Second: How robust is your PCR assay? I prefer DreamTaq Green PCR Master Mix (2X) Catalog number: K1081 from Thermo. This assay applifies everything very robust and you can load it directly on the gel.

Third: This mix has 2.0 mM MgCl2 in the final PCR reaction. But increase it with 25 mM MgCl2 in 0.5 steps and take a look.

With my colleague I have experienced that 0.5 mM MgCl2 can do a miracle on the result. 2.0 mM MgCl2 only a shaddow on the gel when you know where the band should be - 2,5 mM MgCl2 brightest bands!

Fourth: Make a PCR gradient for your gene of interest in parallel with your positive control and different MgCl2 concentrations to find the best PCR setup.

I hope this helps you to genotype your mouse! Best wishes Volker

Thank you everyone I diluted my genomic DNA and have been getting good genotyping results.

Hi Maria Alicai Venegas What were your DNA concentrations before dilution?