Yes, I think PCR or restriction digests will work .

When we work with Arabidopsis knockouts we use a PCR screen to test for homozygosity. Normally we use primers specific to the wild type and primers specific to the TDNA insert used to knock out the gene. If one band is present with the wild type primers then it’s a wild type homozygote, if you have a single band with the insert primers then it’s a homozygous mutant and if you get a double band then it’s a heterozygote.

FISH

To be certain (and probably to get it published) you really have to apply blotting or an 'in situ' technique. Personally, I always use Southern blotting, but of course FISH will work.

PCR is the best choice. To do a gene knockout you must delete an alele integrating an antibiotic gene resistance and the other alele must be replaced by a diferent antibiotic gene resistance cassete. So, to confirm the double integration you should use specific primers designed to target the internal regions of the resistance genes and for flanking genomic regions.

Yes, I think PCR or restriction digests will work .

When we work with Arabidopsis knockouts we use a PCR screen to test for homozygosity. Normally we use primers specific to the wild type and primers specific to the TDNA insert used to knock out the gene. If one band is present with the wild type primers then it’s a wild type homozygote, if you have a single band with the insert primers then it’s a homozygous mutant and if you get a double band then it’s a heterozygote.

But these PCR techniques do not assertain that there is really one copy per diploid genome. qPCR may serve this purpose, but that it is difficult with qPCR to distiguish 50% concentration differences (as required to compare double/single copies per genome).

Southern-blot is a good choice too. But PCR is easier.

FISH for gene knock-out is probably not the method of choice, because the deleted region might be way to small to be detected by FISH, and if I understood the question correctly Thabiso is working with Candida albicans. It might, however, be an option to use a PNA (oligo) FISH probe; still this going to be tricky. I would also recommend to do a PCR.

PCR is the cheapest and easiest method for choice, just performed it as Nicky Creux described. FISH is more intuitive,while southern blot is more dangerous with isotope labeling probe or less sensitive with non-isotope labeling probe.

I am using a recyclable deletion construct (SAT1 flipper) and I have already excised the resisatance gene. Therefore, currently the target locus comprise one copy of the target gene and the flanking sequences which are approx. 600 bp (this is what I have to confirm using one of the aforementioned techniques). I have primers that align downstream and upstream the target gene i.e. they start amplifying from the falnking sequences and I have used these primers to screen for the deletion construct. But now the deletion construct is already recycled so would it be feasible if I use these primers in PCR to screen for heterozygosity and if so should I expect two bands, one representing the target and one representing the flanking regions? This is what I had in mind but I haven't come across a paper with using this approach.

Sure. You are right.

In mice, one can mate the heterozygotes and from the color of the offspring, one can also judge about the status (homo/hetero). Heterozygotes are the choice in the first round because of the issue of lethal gene KOs. Heteros are viable and are then mated for getting homos, if these are viable. This is apart from PCRs of the inserts.

I dont know if it will work for your candida. If it is possible then the new ones can be checked for resistance in vitro to antibiotics/drugs.

I am not in the field though.

First and preliminary  screening is to check the antibiotic resistance to your product. After that go for PCR screening method. Lastly confirm it by making specific probe and do southern blotting.