SNIP SNP MAPPING A MUTATION IN C.elegans
SNIP SNP mapping is a method for determining the rough position of a mutation using snip SNPs (single nucleotide polymorphisms SNPs that also create RFLPs- restriction fragment linked polymorphisms).
For a full description see Wicks et al or the Jorgensen Lab homepage.
Some points before you begin:
You need a mutation that has been backcrossed 4-6 times.
The lab has stock primers for 4 SNPs per chromosome.
All of the lab SNPs cut with DraI making the process easy.
The Hawaiian strain burrows so picking males can be difficult.
Males can be generated, by placing L4 hermaphrodites in 30”C for 3 hours (see specific protocol for details).
Crosses - generating a DNA pool
Obtain a plate of homozygous CB4856 (Hawaiian)
Cross HW males with homozygous mutant mut/mut hermaphrodites L4Õs (2:8, 2:10)
After 24 hours remove the males from the plate or transfer the hermaphrodites to new plates.
Check the mutants/hermaphrodites throw males OR if your mutant has a visible phenotype that the mutant hermaphrodite throws WTs.
Pick 12-20 hermaphrodites to separate plates
After 3-4 days pick Mutants and non mutant progeny either by phenotype or dye filling depending on your mutation.
From the progeny of the plates hermaphrodites pick 40-50 mutant and 40-50 non-mutant worms into lysis buffer.
40-50 worms in 40-60ml lysis buffer + Pro-K seems to work well
If you donÕt have 40-50 worms, 20 worms in 30-40 ml works well also
Cycle on 60-95-10 (see DNA lysis protocol for futher info)
DNA is not stable, use immediately or within a week of being stored at -20”C
You will also need N2, and HW genomic DNA
Set up PCR as for ŌnormalÕ promega PCR
A 10-20 ml reaction is plenty
We have 4 primer sets for each of 6 chromosomes, (24 total reactions)
You will be running N2, HW, Mutant, and WT DNA with these primer sets
Do not try and do all the PCRs at once. Either do each chromosome at a time, or what ever you can handle. There are a lot of different master mixÕs- Mistakes cost money, but going too slow costs time!!
Run 4 ml of PCR out on a 0.8% gel to check the PCR worked (only necessary your first time using the primers)
Digest 6-10 ml of the PCR product
6 ml PCR/DNA
1.5 ml Buffer
water to 15 ml
Digest for 2-3 hours at 37”C, longer or overnight does not help and often makes the products unclear.
Run the digests on a 2% agrose gel (square bottle)
Load the gel from left to right N2 HW Mut WT
Load each chromosome from left to right most – to most +
Load each chromosome in order
Run gel at ~90 v for 40-60 mins
Analyze the banding pattern. Some SNPs N2 cut, and HW does not, and visa versa, did the mutation pick up N2 at any locus?
Take good digital images so you have something to go back to and analyze by computer if necessary.
After you map your mutation to a chromosome using the lab stock primers, you will need to then do 3-factor mapping using visible markers, and/or map using other SNIP SNPs after designing your own primers and SNPs using