Worm Breeder's Gazette 13(1): 22 (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.
SAMPLE PREPARATION-Egg Preps
The Bennett laboratory uses the same protocol Ascaris or Caenorhabditis, although we currently have a lot more experience with Ascaris. We use the hypochlorite solution (40% Chlorox, 50% 1 N NaOH. 10% water) for gravid C. elegans adults or synchronous cultures of Ascaris eggs. When adults have dissolved and eggs have been stripped of their proteinaceous coat, rinse 3-4 times in M9 (or water). Transfer egg pellet to 1.5ml eppendorf tube. Add equal volume of 6:3:1 (methanol:acetic acid:chloroform) to permeabilize vitelline membrane. Invert. Immediately dilute in M9 or water and spin down eggs. Rinse several times. Fix in 4% paraformaldehyde in M9 or PBS (in DEPC water) on ice for 15 min. Rinse 2-3 times. A few embryos should be checked with DAPI at this point to assure they are intact and permeabilized. Mix fixed egg pellet with ~1/3 volume of O.C.T. embedding medium for frozen specimens (Tissue-Tek brand). Freeze the egg mixture on a microtome chuck (or a quarter) which has been pre-chilled on a block of dry ice. These egg blocks are stored at -80°C. until cutting. We have stored blocks for several weeks.
We set the microtome for 12-16 µm sections at -15°C. We fill a slide with sections and let the sections adhere to the slide on a 37°C hot plate for 30 min. After that the slides are fixed two more times in 4% paraformaldehyde (made fresh) in DEPC M9 or PBS + Mg. The first fixation is on ice. The second is at room temperature with the addition of 0.1% deoxycholate and 0.1% Triton-X100. All fixations are done in glass containers which have been baked in the "hot oven"(250°C for >4 hrs.) to remove RNases. Both fixations are for 15 min. The cellular RNA is acetylated in acetic anhydride/triethanolamine solution. (freshly made and used within 30 minutes) at room temp. for 5 min. 100mM Triethanolamine-HCl pH 7.5 250ml
250 ml DEPC-H(2)0
4.64g Triethanolamine-HCL (Sigma T-1502)
560 µI 1ON NaOH
add 625µl acetic anhydride immediately before adding to the slides
Slides are then rinsed 3 times in 1xPBS+Mg or M9 .Slides are dehydrated in 30%,60%,80%,95% ETOH made in DEPC-H(2)0. We air dry them O/N. Store at -80°C until ready for hybridization.
The transcription and hybridization protocols are from Dr. Etsuko Wada, courtesy of Dr. Sandra Petersen, UMC. Some comments may be useful. The lack of any cold nucleotide to supplement the hot label makes the reaction suboptimal in terms of molarity, and causes the probe to be very small (probably 50-75nt). However incorporation is often 95% or better and this small probe doesn't need hydrolysis. Dry down 250 uCi [35S]-CTP in speed-vac (CTP may also be used, cold UTP would then be added.)
1. 2ul 5X Transcription Buffer (we use that which comes with SP6 , T7 or T3 polymerase)
2. 1µl 100mM DTT (Made fresh)
3. 0.5µl RNasin 40U/µl
4. 2µl 1OmM ATP
5. 2µl 1OmM UTP (or CTP when using hot UTP)
6. 2µl 1OmM GTP
7. 1µl Linearized DNA Template 1 µg/µl
8. 0.5µl RNA Polymerase 20U/µl
Gently mix. (Tap tube with finger.) Incubate 30min at 37°C. Add 0.5µl Polymerase and incubate another 30min. Add 89.5µl nuclease-free water. Check % incorporation with PEI chromatography paper. ([35S] count in fluor)
1. 5µl 1 M Tris-HCL pH 8
2. 1µl 1M MgCl(2)
3. 0.5µl tRNA 25µg/µl
4. 0.5µl RNasin 40U/µl
5. 2ul DNase I 1U/µl
Gently mix. (DNase is very labile. Do not vortex.) Incubate 30min at 37°C. Run Sephadex G-50 spin column to remove unincorporated counts.