Hi Hatty,

The first thing to do is indeed Sanger sequencing. However, this must have been done by the facility. Otherwise, you should not know the specific genotype (single bp deletions).

It is also important to know, whether the targeted exon is essential for the function of the protein and, or whether there are alternative transcripts without this exon. Ensembl.org will give you a first idea.

In case that the exon is essential, or no transcripts without this exon exist, a simple Western with any functional verified antibody will do the job. If you are using a commercial antibody, information about the antigen should be available.

Hi Hatty,

For confirmation of KO at protein level you can simply do Flow cytometry with any specific antibody. Because, if a frame shift- mutation is intended, then it is very likely that the protein is not expressed. This you can observe by flow cytometry by presence or abscence of the signal related to the receptor.

Second method is sequencing and I believe there are several companies that do sequencing, which are not that costly at gene level.

Genewiz does it in USA.

You may simply compare the DNA sequences of WT and the KO clones.

good luck,

S

Thanks so much for your help.