Worm Breeder's Gazette 13(1): 28 (October 1, 1993)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have been using the terrific Tc1 -generated deletion method devised by Plasterk; and colleagues (Zwaal et al., PNAS 90: 7431-7435, 1993) to knock-out homeobox-containing genes. We have been able to isolate a handful of deletions in about 8 weeks time. Here we offer some tips that have streamlined the whole process for us.
If your Tc1 strain is not very viable, it helps to backcross it once or twice to get rid of unlinked mutations that cause sickness. Care must be taken to keep the mutator allele. mut-2 maps close to unc-13 (M. Finney, Ph.D. thesis). Backcrossing is also useful because it will decrease the turn-around time by increasing the growth rate.
We have streamlined the screening procedure by eliminating the odious phenol extraction step, and instead prepare a crude Iysate which is PCR'd directly.
ÑSet up for example 100 9 cm plates with two (this could also be 5 or 10) animals each. Let the plates grow until E. coli are almost gone.
ÑAdd 500 µl water, swirl across the plate. Pipette off 100 µI so that you get between 1/5 to 1/3 of the worms in the accumulated liquid*. Keep the plates, put the 100 µl in a 0.5 ml tube.
ÑFreeze the tubes at -70°, put in Iyophiliser to dry (alternatively, you can spin down the worms, suck of some water before freezing, that speeds up drying. If you are careful, you can remove most water and add the Iysis buffer directly without drying).
ÑAdd 25µl single worm Iysis buffer containing ~500µg/ml proteinase K (see Williams et al. (1992), Genetics 131, 609-624), digest for 4 hours at 60 °C.
ÑHeat inactivate at 95°C for 20 minutes.
ÑSet up PCR reactions using between 1/10 and 1/50* volume of worm Iysate. ÑPositives should be visible after a single round of PCR using 35 cycles. Nested PCR is not necessary, as a matter of fact we would have lost some positives since internal primer sites were deleted.
ÑAny potential positives can be checked to see if they are germline events by repeating the PCR from second Iysate prepared independently from the same plate. Alternatively, the primary screen can be done in duplicate by make two independent Iysates per plate.
ÑDivide and conquer by sib selection. (The assay is sensitive enough to detect probably 1/1000 animals, so set up to screen about 1000-2000 animals in the second round, e.g. 100 plates with ~20 animals each)
ÑAs soon as you have a single-worm positive strain, you should start crossing them to
a) make the strain viable
b) to get rid of the mutator allele.
* NOTE: We have been successfully using agar plates, but we had batches of Iysates that did not work well for PCR. Switching to agarose plates and using less Iysate, i.e. 1/20 or 1/50 instead of 1/10, helps . Adding single strand binding protein gp32 can also help for long PCR products)