Because the phenotype you are seeing in some of these mice are similar than previous KO mice, and knowing that your breeders are HETs, you should be able to read the KO band with the ko primers in the breeders. Nowadays the kits to extract DNA from tail clips or ear snips is pretty quick and dirty, I would suggest to extract the DNA of your ear snips in the old way (more time consuming but much cleaner). You will get much cleaner DNA samples and PCR will run smoothy.

Here is the protocol for that: http://jaxmice.jax.org/support/genotyping/tail_phenol.html

In addition, use this software to design new primers: http://www.idtdna.com/Primerquest/Home/Index

I always have a couple of pairs of primers for genotyping so I can easily solve the situation if one of them doesn't work anymore.

Good luck!

Ale

Have you tried to perform a PCR with the KO primers only? In this case you would not have any competition with WT primers and it should work. If it doesn't work you could perform a touchdown PCR with these primers. In any case, you should add controls heterozygous and homozygous mice. In these two controls there will be no mutation in the primer zone, so if this was the case, that is very unlikely, both should be positive. If it does not work you should set up new PCR conditions. Finally, if nothing works you could design another set of primers for KO.

I had a similar problem with my mice. However, the reason was using a high fidelity PCR kit.. the polymerase in this kit required high concentrations of MgCl2 for a better sensitivity. Make sure you are using the right concentration of MgCl2 for your specific enzyme.

Also, in my experience, if it is the first litter that you are analyzing, there is a possibility of only half the litter surviving and thus leading to only WTs and Hets and no KOs.

I would suggest using primers specific to the exon of your target gene that you are knocking out to confirm their genotype.

Because the phenotype you are seeing in some of these mice are similar than previous KO mice, and knowing that your breeders are HETs, you should be able to read the KO band with the ko primers in the breeders. Nowadays the kits to extract DNA from tail clips or ear snips is pretty quick and dirty, I would suggest to extract the DNA of your ear snips in the old way (more time consuming but much cleaner). You will get much cleaner DNA samples and PCR will run smoothy.

Here is the protocol for that: http://jaxmice.jax.org/support/genotyping/tail_phenol.html

In addition, use this software to design new primers: http://www.idtdna.com/Primerquest/Home/Index

I always have a couple of pairs of primers for genotyping so I can easily solve the situation if one of them doesn't work anymore.

Good luck!

Ale

sounds to me like WT DNA contamination in your lab - that would be the simplest explanation. cleaning the pipets only won't alleviate the problem, i had a similar situation many years ago, when making library of a murine genomic locus. since this is most probably aerosol contamination, i'd wipe every possible bench surface (in addition to the pipets) with 70% alcohol and UV irradiate everything on the bench over night.

another remote possibility is to have a genomic rearrangement (recombination) in the KOs that includes and duplicates your WT (or part of it) as well. I dunno your particular locus, but this might be a possibility. a remote primer set should help to clarify this, alternatively MLPA that you'd have to design for the particular genomic region.

I also second doing the PCR separately for WT and KO, including known Hets, WT/WT and KO/KO as controls for both reactions. I will also add that in my experience, I've had more contamination issues using ear clips than tail clips. I think it's because it is easier to clean the scissors (or use a new razor blade) between animals than cleaning the ear clippers. It could just be my own lab superstition, but frankly, I tried ear clipping only for about a month or two, and ended up retaking tails on adults which needs special treatment. At this point, your pups are probably past P21, in which case, you will need to use some form of anesthesia to clip tails, cauterize as usual, and post op analgesia after the tail clipping. It's work, but worth it for good clean genotypes.

Change mutants primers (new design). Perform a separate pcr again.

We usually do hundreds of genotypings per month in our lab with different primers, and occasionally we have a similar problem. It looks like you have a contamination in one of your reagents. It could be ANY of the reagents used to either prepare the DNA or the PCR reactions. I would recommend you change ALL the reagents. You said you changed the mastermix reagents, have you changed the reagents that you use to prepare the DNAs? your problem could be there. Also, ALWAYS clean up bench and pippettes with 70% EtOH before you prepare any PCR. If this does not work, change primers for the WT band (not the mutant) since this is your contaminant. Usually it's not genomic DNA but the PCR product itself that contaminates, especially If the product amplifies really well. Don't worry, it happens to everyone, and it has a solution!

I completely agree with Ines Martinez-Corral.

I have had similar problems with genotyping with some primers suddenly stopping to work for no apparent reason. For a while I actually used separate PCR reactions using primer aliquots from the same stock as before (which used to work in multiplex PCR). After a while I tried the multiplex again which miraculously worked again, I never found out why. But I also regularly order fresh primers as I have noticed that stocks, although frozen, can become less efficient. Also ALWAYS include some control samples (WT, HET, KO mice) so you can at least check if these are still showing up right.

So basically I would suggest to run separate PCR reactions and if that still doesn't work just to order a fresh batch of primers.

Good luck!

Hi thank you everyone for your answers. Just to clarify the WT and KO primers are in separate reactions, so there is no competition between the primers. Furthermore, I always use a known Het sample as a positive control for each reaction (WT or KO) and these have been coming out fine and the water sample is clean.

I have had this struggle for the past 3 years, it comes and goes, yet I have never noticed anything coincidental.

Thank you for your time, it is greatly appreciated.

Kind regards,

Claire

As mentioned above, it sounds like contamination with WT is the simplest explanation. We recently had a similar issue in our lab. We made fresh buffers etc., Do you use filter tips when genotyping? I also suggest making sure the ear clip you use to collect samples is cleaned between mice.

Hello,

I have more or less the same problem. I have perfomed a PCR with my samples and some old ones and I only get bands in the old ones. I have used my samples directly from digestion buffer, other times after phenol-cloroform treatment+ethanol precipitation but no bands.

Suddenly, one sample worked but the other didn't. I have changed my aliquots of buffer, DNA polymerase, primers... I have changed annealing temperature, Mg+2 concentration, adding DMSO... only I got two sample genotyped (I haven't done anything different in those samples). 

Do you have any idea? My mice are at the end of its reproductive age and I don't have hets to cross and very little sample left.

Thanks!

Eva-

I do exactly the same type of genotyping that you do: wt, hetero, homo. I prepare the genomic DNA the old way, have 3 types of oligos for each allele, and I am extra-careful when I get the ear snips. When I find the wt band in all my samples (something that acassionally happens to me), I switch primers and the PCR is fine then. Also check different dilutions of your template: this works very well.

It would be nice to verify specificity of primers by sequencing of amplified product.

I have the same praoblem with you, I am genotyping mouse tail and I looking for hetero mice (+/-) (3 bands) but the band is all WT (1 band). I used the same primer from my Senior, and she never have this kind of trouble. How do you solve your problem??