primers for mod-1

Hi Everyone,

I need to genotype a number of strains that may or may not have mod-1. I haven’t done any molecular biology in a while. Does anyone know
what primers I can use for mod-1? Thanks!

I usually just take the sequence of the first 20 base pairs at the start of the gene for forward primer, then the reverse sequence of the last 20 base pairs at the end of the gene for reverse primers. Primers targeting the promoter and UTR regions have higher chance for non-specific bindings, but PCR that covers a little upstream and downstream of your gene is useful if you want to sequence the PCR product (to look for point mutation etc.). You can always paste your primers to the BLAST tool in Wormbase to check for non-specific bindings.

After choosing the primers, check them for some technical parameters (GC 40%-60%, no more than 5 runs or repeats, preferably 2-3 G/C in the 5 bp at the end of the primer), then PCR with single wild-type worm (N2) lysate with a variety of annealing temperature settings and choose the best one for the mutants or screening. If all the PCR conditions present multiple bands, consider using a different primer(s).

Good luck!

The interface is a bit clunky but I’ve always had good results using primers found for me by Primer3.

Do you mean they might have random mod-1 mutations or mod-1 gene sequences? Or do you mean you have a bunch of strains that may or may not have a specific mod-1 mutant (like ok103)? If so, then you want primers that will allow you to detect ok103, not just mod-1.

Sorry, I should have made it clearer. The constructs I’m interested in may or may not have mod-1(ok103).

I had a little bit time of time this morning and made an ApE file for you. Use your favourite program or download ApE. Double check that I got the ok103 lesion right. I labelled it in pink. If you use the primers I labelled in the file, wt should amplify a ~5kb fragment and ok103 should give 891bp.

One addendum to Snug’s comment: it’s not enough to have primers to test for the presence of the deletion, you also need primers to test for the presence of the wild-type allele. They’re usually not the same primers, because it’s harder to amplify a much larger product, and if you PCR from an ok103/+ heterozygote using primers to detect the ok103 deletion you would likely not get a wild-type 5 kb band, because the smaller product would amplify so much more efficiently (5 kb is large enough that you might not get a product amplifying from the wild type, even in the absence of the ok103 mutation).

It helps if you choose the primers for detecting the wild-type allele so that their product is a different size from that given when the other primers successfully detect the ok103 deletion. This lets you run the PCRs from a given worm or plate in one lane, or even do the PCRs together in the same tube (something I’ve never done, but I know others do successfully).

Thank you all for your help!

Piggybacking on Hillel’s comment. Yes, make a 3rd oligo for the wild-type gene that gives a band distinguishable from the mod-1 deletion PCR!!! I try to make the wt PCR smaller than the mutant so that if you fail to see the wild-type band and a clear mutant band, you’re sure it wasn’t too big to PCR. A 5Kb PCR would be way too big, as I imagine it would fail through standard wormlysis PCR genotyping 99% of the time. I prefer one band at or above the 500 bp marker, one just below, say 300 bp, and run a 2% gel. And yes, put all the oligos into one PCR and run wild type and mod-1 mutant control animals next to all of them (I typically do two of each per genotyping run).

We do tons of genotyping PCR on deletions. 1) We always do triplex (two primers flanking the deletion and one inside the deletion), 2) We always keep the key products at 500 bp or less, and 3), as noted, keep the wt band band smaller than the deletion band, and 4) always run controls: +/+, m/+, m/m ( unless the m/m i animal is sterile or lethal).