Hi Abrar Ahmed,

Depending on your mutation you may be able to identify het/hom/wt by using gel electrophoresis. If your mutation is for example a 50bp deletion, simply performing a PCR and running the sample on a 1% agarose gel will enable you to distinguish between wt (one strong band appearing at the normal size), heterozygous (one band at the wt size and one band at the mutant size) and homozygous (one strong band at the mutant size). I have managed to resolve deletions of 10bp on 4% agarose gels in the past pretty quickly. However if your mutants have a small deletion/insertion or base pair substitution you may have to see if there are any restriction enzymes that cut only one of the mutant or wild type sequences. This will allow you to digest the PCR product and depending on the subsequent bands that appear on the agarose gel, enable you to determine the genotype. This website is useful for finding restriction enzymes that may cut your sequence: http://www.restrictionmapper.org/ I would advise confirming this with sequencing for the first time to ensure what you are seeing on the gel corresponds to the actual sequencing results. But once up and running it is a cheap and efficient way to genotype zebrafish.

I hope this is helpful,

Ben

Hi Abrar Ahmed,

Depending on your mutation you may be able to identify het/hom/wt by using gel electrophoresis. If your mutation is for example a 50bp deletion, simply performing a PCR and running the sample on a 1% agarose gel will enable you to distinguish between wt (one strong band appearing at the normal size), heterozygous (one band at the wt size and one band at the mutant size) and homozygous (one strong band at the mutant size). I have managed to resolve deletions of 10bp on 4% agarose gels in the past pretty quickly. However if your mutants have a small deletion/insertion or base pair substitution you may have to see if there are any restriction enzymes that cut only one of the mutant or wild type sequences. This will allow you to digest the PCR product and depending on the subsequent bands that appear on the agarose gel, enable you to determine the genotype. This website is useful for finding restriction enzymes that may cut your sequence: http://www.restrictionmapper.org/ I would advise confirming this with sequencing for the first time to ensure what you are seeing on the gel corresponds to the actual sequencing results. But once up and running it is a cheap and efficient way to genotype zebrafish.

I hope this is helpful,

Ben

Thank you sir for explanation Benjamin Munro

I am using two mutants with 5 and 1 base pairs deletion. I think i cannot use gel for confirmation. i am gonna try option third.

Best regards

Abrar Ahmed

Hi Abrar Ahmed ,

Just to avoid confusion, if you find a suitable restriction enzyme that cuts either your WT sequence or mutant sequence, you will still need to run the digested PCR product on a gel to see your results.

Many thanks,

Ben

Yes sir Benjamin Munro

I understand from your first message that after using enzyme we have to check mutant and wild type bands

Thanks for explanation.

Best Regards

Abrar Ahmed

Hello

Take a look

Article Identification of Genetic and Chemical Modulators of Zebrafi...

Good Luck

Thank you very much Professor Saleh Alkarim and Elizabeth E LeClair

I agree with Elizabeth that A1 bp so difficult to delete

For 1bp alterations you can use a taqman assay if you going to use your fish a lot. It's about 2 euro per sample in total. So I guess using a restriction enzyme is still the cheapest.

Yes sir I am using continuously Zebrafish and for confirmation of mutants we send sample for sequencing.

we also use restriction enzyme but for confirmation we send some of them for sequencing too. Than you very much sir @Bart Appelhof.