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[
Worm Breeder's Gazette,
1998]
In case of weakly expressed genes, Northern blot analysis may not be sensitive enough to detect transcription unless poly(A)+ RNA is prepared. We would like to report a simple method of RT-PCR assay from single animals that does not require poly(A)+ RNA preparation. Washed with M9 buffer, each single animal is transferred in 2 ul of TE (pH 8.0) in a 0.5 ml tube and frozen (-80oC, 20 min). 1 ul of lysis buffer [0.5 mg/ml proteinase K in 10 mM Tris (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 0.45% Tween 20 and 0.05% gelatin] is added and mixed well. After the addition of a mineral oil overlay, the tube is heated at 65oC for 1 hour and the reaction is stopped by the boil for 15 min. After cooling to 4oC, 1 ul of DNase I (7.5 units/ul, Pharmacia Biotech) is added and incubated at 37oC for 30 min. The reaction is stopped by boiling for 15 min. 46 ul of a master mix is then pipetted on top of the mineral oil overlay. The mix is formulated to bring the reaction volume to 50 ul using DEPC H2O with these final conditions: each 10 pmol of sense and anti-sense primers, 25 ul of 2x reaction mix and 1ul of SuperScript II RT/Taq DNA polymerase mix (GIBCO BRL). After a brief microfuge spin, the sample is incubated at 45-55oC for 30 min for cDNA synthesis. Then, 94oC for 2 min followed by amplification of 45 cycles of 94oC for 30 sec, 50-65oC (depending on the primer set) for 30 sec and 72oCo for 0.5-3 min (1 kb/min), followed by 72oC for 5 min. The PCR product (10 ul) is analyzed on 0.8-1.5% agarose gels. Using this method, we have been able to detect a typical
sod-3 RT-PCR product of which the expression is lower than that of other genes. To avoid error in different stages, we recommend using some animals at the same developmental stage and duplicate with each primer set. This system is also useful for late-stage embryos. References: 1. Williams, B., D. et al. (1992) Genetics 131: 609-624 2. Andachi, Y. et al. (1993) WM'93: 33 3. Lee, E. H. et al. (1997) Focus 19 (2): 39-42 4. Sitaraman, K. et al. (1997) Focus 19 (2): 43-44
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[
Worm Breeder's Gazette,
1990]
The accompanying diagram shows the system which we use to pressurize our microinjection setup. The key feature of this system is the three way ball valve through which pressure at the needle is applied and then rapidly relieved during each injection. Pressure must be off between injections because the high flow associated with optimal injections will float animals off the pad before the needle can be inserted. It may be possible to adapt your current system to include such a valve, or to construct a similar system with other parts. [See Figure 1] 1) Leitz microinstrument collar (part no. 520145). Holds the injection needle. 2) 3/32' outside diameter (1/32' I.D.) Tygon tubing. This tubing, once stretched, will fit snugly down the opening in the back of the microinstrument collar. This junction is taped with electrical tape. The tape keeps this joint from getting worked loose during the frequent handling of the microtool collar. The tape should not be necessary to make the pressure seal. 3) 21g metal tube (cut off of a syringe needle). Fitted halfway within the 3/32' tubing, the protruding portion is inserted into the 1/16' tubing and serves as a connecter. This connection is also wrapped with electrical tape. 4) 1/16' polyethylene tubing (rigid plastic or nylon tubing works best with the Swagelok adapters). Note; if you plan to HF treat your needles (Worm Breeders Gazette 11-1:18), then this tubing should be long enough to reach a nearby dissecting scope. S) Swagelok reducer (part no. B-100-R-2). Connects the 1/16' tubing to the Swagelok 1/8 connector which comes with the three way valve. 6) Swagelok three way ball valve (part no. B-41XS2). Should be mounted or clamped in place. This valve is used during each injection to apply pressure at the needle. It should be placed within easy reach but should not be on the vibration free table. 7) Swagelok male connector (part no. B-100-1-4). This connector threads into the 1/4 NPT female socket of the STREET TEE. 8) CAJON STREET TEE connector (part no. B-4-ST). Male end threads into the pressure regulator. Female ends accept the tubing connector and the pressure release valve. 9) Swagelok toggle operated shut off valve (B-1GM4-S4). This valve opens the system to allow a quick release of pressure. This is necessary when using the pressure regulator to make downward adjustments in pressure, for example if there is too much flow at the needle. 10) Pressure regulator. Must have a 1/4 NPT Female thread in order to accept the STREET TEE described above. Allows the pressure to be increased or decreased. Pressures required for adequate flow range from 10- to 100-lbs/sq.in. depending on the needle; higher pressures tend to blow connections or to propel the needle out of the holder at high velocity. 11) Nitrogen tank. Should be positioned so that the regulator handle can be reached easily without getting up during injection. Happy injecting!!
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[
Worm Breeder's Gazette,
1981]
A 4 liter glass 'fermenter' has been used instead of Roux bottles in our lab for C. elegans mass liquid cultures. It is made of glass plates stuck together with silicone rubber. It is autoclavable at 120 C, and provided with an aluminium cover containing two noles, into which tubes, are sealed by silicone rubber. One tube (the shorter one) includes a 'weighted' valve, supplied with a movable but hole- covering glass ball. The other an inverted T, the horizontal arms of which are perforated with holes, the prefiltered condensed air entering via the vertical section. This apparatus is practical for both bacteria and C. elegans. New cultures are started by transferring the cover to a new, sterilized culture from an old one. [See Figure 1]
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[
Worm Breeder's Gazette,
1996]
The following protocol uses TRIZOL reagent from Gibco-BRL to isolate RNA from whole worms. TRIZOL reagent is a solution of guanidine isothiocyanate and phenol which simplifies the original method published by Chomczynski and Sacchi1 and can be used to isolate RNA, DNA and proteins from the same sample. The following protocol does not involve sonication and results in good yields of RNA which have been used in our laboratory for Northern analysis, RT-PCR, RACE reactions, and cDNA synthesis. For 2mls of worms grown in liquid culture: 1. Add 8mls of TRIZOL reagent to 2mls of packed worms in a 15ml polypropylene tube. Vortex and invert tube to solubilize and lyse worms. {NOTE: Getting the packed worm pellet into solution can take some time and vigorous vortexing. This is fine because the longer you let the worms sit in the TRIZOL, the better the lysis and thus more RNA will be isolated. Letting the solubilized worm pellet sit in TRIZOL for at least five additional minutes is recommended. At this step the solution should be pink and will contain thread-like insoluble material.} 2. Split solution into 8 epi-tubes. About 1250ul per tube. 3. Incubate at RT for additional 5 min. Spin 14K for 10min at 4oC in a microfuge to remove insoluble material. 4. Remove liquid to a fresh epi-tube. Add 200ul CHCl3 to each tube. 5. Invert/Vortex for 15sec. Let incubate at RT for 2-3min. {NOTE: I found with the guanidine isolation that the 15sec vortex is critical. Too little vortexing at this point can result in "smeary" RNA preps. I have no explanation for this.} 6. Spin 15 min. at 14K at 4oC to separate phases. 7. Remove upper aqueous phase to a fresh tube. Add 500ul isopropanol and mix. 8. Incubate 10 min. at RT to precipitate RNA. Recover by spinning at 14K for 10 min. at 4oC. 9. Carefully remove aqueous away from pellet. {NOTE:Pellet will be very white.} 10. Wash pellet with 100ul of 75% EtOH in diethyl pyrocarbonate (DEPC) treated H2O. Vortex briefly. {NOTE:Pellet will often float free. Also RNA pellets can be stored in the 75% EtOH at -80oC for up to one year safely.} 11. Spin at 7.5K for 5 min. at 4oC. 12. Remove supernatant and air dry pellets for 5-10 min. 13. Vacuum dry pellet for ~7 min. WITHOUT centrifugation. {NOTE: Extensively drying RNA pellets makes them hard to resuspend.} 14. Dissolve pellets in 25-50ul DEPC-H2O. To help dissolve heat at 60oC for 10 min. {NOTE: If RNA won't resuspend completely you may have a good yield and more DEPC-H2O can be added. I have often had to add from 200ul to 500ul to get a good resuspension. 15. Dilute 1-5ul of prep into 1ml of dH2O and take OD 260 & 280 to determine concentration and purity. {NOTE: An A260/280 ratio of indicates partially dissolved RNA.} My yields have typically been between 1-4mg of total RNA from 2mls of whole worms.
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[
Worm Breeder's Gazette,
1997]
I have developed a fixation protocol which provides an alternative set of conditions to test antisera that work poorly on paraformaldehyde fixed worms (e.g. the Finney protocol). A Bowin's fixative of 75 ml saturated picric acid [real nasty stuff- explosive in crystal form!], 25 ml of formalin, and 5 ml of glacial acetic acid is made. Many fly labs use this fixation solution which may be stored at 4=B0C for many months. Nematodes grown on NGM plates are washed off the plate using 2 to 3 ml of H2O and spun down in a glass 12 ml conical tube in a clinical centrifuge at room temp. The supernatant, save 50 ul, is removed by aspiration. A mix of 400 ul of Bowin's, 400 ulmethanol and 10 ul of BME is placed in the glass tube to initiate fixation and subsequently the worms are transferred to an eppendorf tube using a glass pipette (600:200:10 Bowin's: MeOH: BME also works). The tube is rocked on a Labquake shaker at RT for 30 minutes, then quick frozen in liquid N2. The tube is quickly thawed under running hot water until the solution melts (but before it warms up past RT). The tube is rocked an additional 30 minutes. A solution of BTB [ 1X Borate buffer, 0.5% Triton X-100, 2% BME] is made from 50X borate buffer ( 1.0 M H3BO3, 0.5 M NaOH). The worms are spun from fixative in a centrifuge [ 2-3 seconds in an eppendorf 5415C ]. The fixative is aspirated, and 1.4 ml of wash solution is added, rocked a few seconds to mix. Spinning and aspiration completes the first wash. Two more washes are done, and the worms are then resuspended again in 1.0 ml BTB ( in the lower volume worms move more during rocking). The worms still have a yellow tinge due to the picric acid at this point. The worms are now rocked for approximately 1 hr at RT. The speed at which the yellow picric acid destains is a good sign of how well the worms are going to stain. If the worms remain yellow after an hr of washing, they usually end up impermeable to antibodies. Wash again with BTB and incubate an additional 2 or 3 hrs. Wash with BT (BTB lacking BME) one time, and AbA (1X PBS, 1.0% BSA, 0.5% Triton X-100, 10 mM NaAzide) solution two times. Incubate 30 minutes or more then begin standard Ab incubations. I never switch tubes during this procedure. I store these worms at 4 C and they often stain relatively effectively a month later (and sometimes they don't). In my hands this procedure gives good staining in greater than 90% of the worms. Our antibodies to RAB-3 and SNB-1(synaptobrevin/VAMP) work much better on Bowin-fixed worms than on well permeabilized 'Finney protocol'-fixed worms. UNC-64 (syntaxin) Ab works equally well in the protocols, and SNT-1 (synaptotagmin) Ab works much better using the Finney fixation than the Bowin's fixation, although staining is still reasonable in Bowin's. Thus, Bowin's fixation provides an alternative condition that may reveal different epitopes on proteins. I have not altered fixation length very much, but I have found that increased fixation does reduce permeability. A 1 hr fixation seems to be a good compromise between fixation length and retaining permeability, but I would suggest reducing fixation to 30 minutes if you find your worms are not permeable when you try the procedure. I am relatively certain that small changes in the temperature during fixation can cause dramatic changes in the extent of fixation.
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[
Worm Breeder's Gazette,
1984]
In our efforts to make monoclonals against the levamisole receptor, we've needed a passel of worms. Which means growing a helluva alot o' bacteria. 250 liter fermentors do not seem to abound in these parts and so we're unable to emulate the Johnson-Rand gang (1.5 kg of worms from a 250 L fermentor). We've turned to making the best of our Lab- Line S.M.S. fermentor (see an earlier article by S. Ward and E. Hogan, WBG 5 2, p.10). Using pure oxygen and a slightly modified version of the SLBH medium of Sadler, Miwa, Maas and Smith, Laboratory Practices 23, p.642, 1974, we've been able to grow 260 gm of E. coli per 3.3 liter of medium in a 7 hour fermentation run with very good reproducibility. A simplified version of the method described by Sadler et al. works just fine. We use all El Cheapo chemicals from Sigma for our SLBH medium. If you follow this method, be very careful with the oxygen. As you should know, it makes things highly combustible. Apart from the cost of the regulator and a tank rental charge, 244 cubic feet of oxygen cost us only $6.40 and it lasts 4 or 5 runs, giving us an average of 80 gm more a run than the 180 gm yields described by Ward using air. [see Figure 1] Use of the Sigma chemicals leads to a precipitate in the SLBH medium but otherwise things work just fine. To start things off, a 300 ml overnight culture is grown at 37 C in SLBH. We use E. coli strain NA22 whose genotype seems to be lost in the Dark Ages but the genotype does include a ts lethal so careful temperature control is important. The next morning the S.M.S. high density fermentor containing 3 liters of the complete SLBH medium is inoculated with the overnight culture and run at 320 rpm with an initial O2 flow rate of 1-2 liter per minute at 1 or 2 psi. Unlike the method of Sadler et al., all the glycerol needed is present from the start. Also, to set up a run, we autoclave the growth chamber and media separately (partially necessitated by the small size of autoclaves in Missouri). The growth chamber bottle is autoclaved lying on its side in a sling made from Tom Sanford's car seat belts. Another useful invention of Tom's is that the exiting gas from the fermentor is directed down a drain and up the sewer side of the trap. Such an exhaust tube goes a long way to making you more socially acceptable in your building. After 3 hours into the fermentor run, the O2 flow rate is turned up to 4-5 liters per minute and left at this rate for the remainder of the run. Around 4 hours, a slow trickle of cooling water is turned on to cool the hot little growing bugs. One advantage of the S.M.S. fermentor is the bacteria produced are in such a small volume that they can easily be collected in about 10 sterile 500 ml centrifuge bottles. Getting worms from the bacteria. We've found Nalgene transparent polycarbonate carboys to be excellent worm growth chambers. As advertised, they are lightweight and durable but somewhat more expensive than glass (~$40 from VWR). Due to our puny autoclave size, we've had to cut most of the necks off our carboys and create makeshift caps out of the lids of Sigma kilogram bottles. Two holes are cut in a lid as entrance and exit ports for 3/16 inch by 1/16 inch by 5/16 inch Tygon tubing to carry air into and out of a carboy. A third 3/4 inch diameter hole is cut as a sampling port and plugged with a #3 silicone rubber stopper. To keep the water level up, sterile water is also added through this port during worm growth (we don't think pre-humidifying air is good for the 'sterility' of worm cultures). The Tygon tube bringing air into the carboy is run to the bottom of the carboy where it is held weighted by stainless steel nuts loosely held onto the tubing by inserting a flanged plastic tubing adapter into the end of the tubing. The exit tubing begins just inside the makeshift lid and both inlet and exit tubing are glued to the lid by epoxy resin. Air coming in through the inlet tube is filtered through sterile cotton packed in a gas drying tube. Carboys are autoclaved filled with 16 liters of the modified S medium described by Ward (op cit). E. coli are added suspended in S medium to give a final concentration of 28 gm/liter of bugs and a final volume of 18 liters in the carboy. Before inoculating with worms, a carboy is vigorously bubbled for 24 hours. Aeration should be violent enough to form a standing wave 1 to 2 inches high. We have otherwise proven a few times too many that the worms will die of anoxia. The inoculum consists of 600 ml of a clearing culture of worms grown at a similar bacterial concentration in a 2800 ml Fernbach flask by rotary shaking. With such an inoculum, the carboy clears in about 4 days and becomes mostly dauers about a week after that, yielding 100 to 130 gm of dauers from ~500 gm of E. coli. Carboys are sat in an ice-water filled sink for 2 hours and then allowed to continue settling overnight at 4 C. After siphoning off the liquid, settled worms are cleaned by sedimentation through 14% w/w sucrose and flotation on 30% w/w sucrose in 250 ml centrifuge bottles spun at 2000 rpm in a Sorvall GSA head at 4 C. Sucrose is employed because on this scale, Ficoll 400 would be prohibitively expensive. For regular stages of worm, 35% w/w sucrose probably could be substituted in the flotation step with some loss of worms to the bottom of the bottle. A yield of over 200 gm of worms per carboy should be obtainable if the worms are not allowed to go to dauers. As for fermentor runs, directing the exhaust air from worm carboys down a drain helps make the air in the lab breathable. Mixing some air in with the pure oxygen from fermentor runs is probably also a good idea to avoid accumulating an oxygen atmosphere in your local branch of the sewer.
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[
Worm Breeder's Gazette,
1988]
Borrowing yet again from yeast molecular biology, the following procedure has been used to isolate clean, intact RNA from small quantities of worms. This saves the time and effort of growing up, and grinding in liquid nitrogen, large liquid cultures. This procedure should make it possible to process easily 10 to 20 stocks in parallel for screening by Northerns. Yields are ~200 g of clean RNA from 4x9 cm 'high yield' plates (see below), or about 2 g of clean RNA from a single, 5 cm streaked plate. Mini-prep. Wash worms off 4 plates (5 cm) that are just clearing. If you want, the worms can be incubated in S-media for 30 min to digest whatever they have in their gut, and then purified by standard sucrose flotation in a 5ml tube. Resuspend the worm pellet in 0.5 ml of liquid. (Use any RNAase-free, solvent resistant, sterile tube that is big enough. I use Falcon 2098 50ml disposable polypropylene tubes.) Add 2ml GuEST buffer and 2ml PCI (see below), and then 6g glass beads. Vortex on high speed, using a regular bench vortex, for 2 min at room temp. Draw the liquid off the beads with a pipetman into 4x1.5 ml eppendorf tubes, rinse the beads with 0.5ml of PCI, and add this to the microfuge tubes. Microfuge for 2 min. Transfer the aqueous layer to fresh tubes, add 1/10 vol 3M NaAcetate, pH 6.0, and re-extract with PCI. Spin, transfer the aqueous phase (leaving the interface, if any, behind), and precipitate the RNA by the addition of 2 vol of 100% ethanol. -20 C for 20 min, spin in microfuge 10 min, decant, and wash pellet with 80% ethanol. Dry, and dissolve the pellet in 0.3ml of dddH20 (see below). Add 0.9ml of 4M NaAcetate (diethylpyrocarbonate ( depc) treated), and let sit at least 5 hr at 4 C. Spin in microfuge 10 min, discard supernatant. Dissolve the pellet in 0.1ml dddH2O, add 5 l 3M NaAcetate, and 210 l 100% ethanol. Precipitate at -20 C for 20 min, spin, wash with 80%, and dissolve the final pellet in 50 l dddH2O. Dilute 2 l to 1ml with dddH20, read A260 and multiply the absorbance reading by 20 to get the RNA concentration in mg/ml. p(A)+ selection, using poly(U) Sepharose (Jacobson [1987] Meth. Enz. 152:254261), should give 2-4 g of p(A)+ RNA, although using total RNA has given me good signals on a Northern with an actin probe. If you are in a hurry, you can skip the NaAcetate precipitation step, since large MW DNA won't transfer upon Northern Blotting. Micro-prep. Wash the worms off a single 5 cm plate that is just clearing. Repeat the above procedure, using 50 l worms, 100 l GuEST buffer, 100 l PCI and 0.5g glass beads. Rinse beads with 50 l PCI. Do the salt precipitation of RNA in 30 l by adding 90 l of 4M Na Acetate, and dissolve the final pellet in 10 l. Frozen Worms. I have used this procedure, successfully, to prepare poly(A)+ RNA from frozen worms. Washed worms were suspended in 0.1M NaCl, quick-frozen in liquid nitrogen, and kept at -70 C for 1 month. Use 4ml of GuEST, 4ml of PCI and 12g of beads per ml of worms. Thaw the tube just until the frozen plug of worms can be removed, and drop the plug into the GuEST/PCI. Proceed as with the mini-prep, although you may want to vortex a bit longer. Buffers. GuEST (From M. Goedert) Add 245ml sterile distilled water to 200g Guanidine isothiocyanate (BRL Ultra Pure). Add 21ml 1M Tris pH 7.4, and 42ml 100mM EDTA. Heat gently to dissolve. Add 9ml Sarkosyl, and 4.2ml -mercaptoethanol. Bring volume to 420ml with sterile distilled water. Filter through a sterile Nalgene filter, and store at 4 C. PCI. Phenol:Chloroform:Isoamyl alcohol, 25:24:1 Glass Beads. 0.3 to 0.4 mm diam (although I haven't tried other sizes). I borrowed mine from J. Kilmartin, but apparently BDH and Sigma sell them. They can be acid washed, baked and re-used. dddH20. Add depc (0.07% v/v) to sterile, double-distilled water, shake for 10 min, and then autoclave. 'High Yield' plates. (From A. Spence) Add a drop of 20% glucose to a large NGM plate, and then spread the plate with a wild-type bacteria such as NA22. Let sit overnight before adding worms. HINTS: Wear gloves, use only sterile tubes and pipette tips, and generally treat the RNA as you would HIV, and you should have no problems. A quick method for checking the quality of your RNA is on a 0.8% agarose minigel. Denature the RNA for 10 min at 65 C, chill on ice and add sterile glycerol/dye/buffer. Run 2 g RNA/lane in a RNAase free gel box with standard gel buffer, ~7 V/cm for 40 min. EtBr stain 5', photograph. You should see two tight, rRNA bands, with no high MW DNA visible. Avoid using old plates, where the worms have started burrowing, as the softened agar tends to wash off with the worms, and contaminate the RNA. In these cases a small-scale sucrose flotation might be useful to clean up the worms, but I haven't yet tried this.
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[
Worm Breeder's Gazette,
1992]
As Dr. R. Horvitz's laboratory asked us for a good protocol for RNA extraction from Ascaris eggs and embryos, other labs might also be interested in this method. The problem with Ascaris is that it has a very rough egg shell; that's why it is rather difficult to obtain RNA from different developmental stages. With the method described below, one can isolate it efficiently. This RNA can be used to perform Northern blots, polyA+ isolation, c-DNA cloning, etc. Collection of eggs from Ascaris lumbricoides var. suum -Decomposition of the uteri and removal of the proteinatious layer of the eggs are done by incubating the uteri overnight at 4 C in 0.5 N NaOH with gentle agitation with a magnetic stirrer. -Let the eggs settle down (ca. 1 hour) or centrifuge them in a swing-out rotor at 7009 for 2 min. Rinse the eggs 5 times in 10 volumes of tap water. At this stage, the eggs are resuspended in 0.1 N H2S04 and can be stored at 4 C for days, months or even years! To obtain different embryonic stages, one incubates the eggs for the desired time with 10 volumes of 0.1 N H2 SO4at 30 C in a large erlenmeyer; the latter, closed by a paper towel, is gently agitated in a rotatory shaker. Removal of the chitinous layer -Resuspend a pellet of eggs in 5 vol. 3.8% NaClO during 1 min. in a 50 ml Falcon tube. -Centrifuge in a swing-out rotor at 7009 during 1 min. -Resuspend the eggs in 5 vol. 3.8% NaClO by inverting the tube 3-4 times. -Leave the tube in a vertical position for 10 min. The eggs should now swim on top of the solution. -Add 1 vol of water and mix. -Centrifuge in a swing-out rotor at 7009 during 1 min. -Rinse the eggs 5 times in 10 volumes of distilled water. Note: between the second and third rinse the non-fertilized eggs burst, giving a white color to the water. RNA extraction: This method is based on Chomczynski's protocol (Analytical Biochemistry 162 (1987), 156-159) -Resuspend the eggs (ca.0.7 ml of packed eggs) in 3 ml of solution D: 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol. -Homogenize the eggs in a teflon-glass homogenizer with 1 mg of glass beads (diameter 0.1 mm); 3-4 strokes are sufficient. -Check whether the lysis of the eggs is complete with a microscope -Transfer the solution into a 15 ml Falcon tube. -Add 0.3 ml of 2 M NaAc (pH 4) and mix. -Add 3 ml of water-saturated phenol and mix. -Add 0.6 ml of CHCl3 /Isoamylalcohol 49:1. Mix vigourously during 20 sec. -Chill on ice for 15 min. -Centrifuge for 20 min. in a swing-out rotor at 10 0009 at 4 C. -Resuspend the pellet in 0.7 ml of solution D. Heating at 68 C and vortexing make the resuspension easier. -Transfer the solution into a sterile Eppendorf tube. Precipitate 1 hour at -20 C with 1 volume of isopropanol. -Centrifuge for 15 min. at 4 C. -Wash with 1 ml of 70% ethanol. -Dry the pellet at 68 C. -Resuspend the pellet in 0.1 ml RNAase-free water at 68 C. By using this method, one should obtain a total RNA concentration of about 1 mg/ml from 0.7 ml packed eggs.
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[
Worm Breeder's Gazette,
1985]
Worms must be frozen slowly, if a large proportion of them are to survive. Unfortunately, they sink to the bottom of the tube while freezing, so that the entire tube must be thawed to get them out. Thus, depending on the compromise between freezer space and strain security chosen, a strain must be regrown and refrozen every few times it is thawed, exposing it to hazards of spontaneous mutation, confusion with other strains, or outright loss. I have been freezing worms in 0.2% agar. The agar supports them while they freeze, so that one need only scrape out about 0.1 ml of ice with a spatula to thaw them. The agar seems not to affect survival, but I have only a few months experience, and the sickest strains I've checked have been CB190=
unc-54(
e190), and CB128 =
dpy-10(
e128). (The latter is said to be difficult to freeze.) Protocols are identical to Mark Edgley's (WBG 8(3): 9798) up to the actual freezing step. Then I cool the worms suspended in liquid to 0 C, add an equal volume of S medium + 30% glycerol + 0.4% agar, melted and cooled to 50 C beforehand, vortex, aliquot (0.2% agar is soggy enough to pipette), and freeze in styrofoam blocks as usual. To thaw some worms, I sterilize a pointed spatula with flaming ethanol, scrape out about 0.1 ml of ice with it, and dump the ice in the middle of a 6 cm NGM plate spread with OP50. I put 1.8 ml in a tube rather than the 0.6 ml that Mark uses, so that each is good for 10-20 thaws. Naturally, this trick is most useful when one has many L1's and L2's to freeze, since only a small volume of ice need be thawed to get some survivors. I grow my worms on 10 cm enriched plates, which contain 0. 5% peptone (double that in NGM) and 0.1% yeast extract, in addition to the usual ingredients of NGM. The extra goodies support a dense bacterial lawn, and therefore a large crop of worms. I also use 3% agar in these plates (rather than the usual 1.7%), which somewhat reduces burrowing.
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[
Worm Breeder's Gazette,
1992]
We have begun DNA sequence analysis of 50 recessive
mec-4 mutations. By combining aspects of a few different protocols we have come up with a method for sequencing of PCR products that is reliable and gives high quality data. We hope that this protocol may be of use to others involved in sequencing projects! A. Generation of the template DNA for sequence analysis 1. Amplify genomic DNA in standard PCR reaction. We use 2 g genomic DNA and approximately .5 g each primer (our primers average 21 bp in length) in 100 l reaction volume. 2 Run an agarose gel to assay the concentration of amplified DNA. Ideally, the concentration of DNA should be at least 1 g in 10 l. If the DNA concentration is very low, either ethanol precipitate to concentrate it for the next step or reamplify. Also if large visible amounts of primers remain, it is good to remove them as outlined in Step 4 below. 3 Amplify one strand of DNA. In separate experiments we have used either a new primer internal to the original boundaries of the PCR product or we reused on of the original primers for the single stranded amplification. Both approaches work quite well. Usually we use 10 l of the DNA generated in Step 1 above (About 1 g if possible) and about 200 ng of primer in 100 l volume. 4. Purify the ss amplified DNA away from unincorporated primers by passing over Miniprep Spun Columns form Pharmacia. Alternatively, make homeade columns with Sephacryl S-400. 5. Check DNA concentration on an agarose gel. For certain success, about 2 g of single-stranded DNA is used in each sequencing reaction. If the DNA concentration is too low, DNA can be ethanol precipitated to concentrate the sample or a second single-stranded amplification step is suggested. The amount of DNA template is very important. B. Denaturation of the template and annealing of sequencing primer 1. Add 8 l of template DNA and 1 l of 1 M NaOH to a 1.5 ml microcentrifuge tube; incubate at room temperature for 10 minutes 2. Add 1 l of 1 M HCl to neutralize the solution and then immediately add 2 l (about 200 ng) of sequencing primer (from the complementary strand, of course) and 2 l of sequencing buffer (as in Sequenase kit from US Biochemical). Note: perform this step on each tube individually, ie., if you are doing 10 reactions do not add HCl to each tube and then go back and add primers. Neutralize the first tube and add the appropriate primer and buffer and then start the next tube. This is important. 3. Incubate at 37 C for 15 minutes to allow the primer to anneal to template DNA C. Sequencing reactions Reactions are according to instructions in the USB Sequenase kit with a few changes: Labelling reaction: -14 l annealed DNA (the entire reaction prepared above) -1 l DMSO (added fresh) -1 l DTT. 0.1 M -2 l diluted dITP mix (1:5 dilution) -0.5 l 35S -2 l diluted sequenase* *When diluting Sequenase for reactions the dilution mix is made as follows: enzyme dilution buffer, 6.5 l pyrophosphatase, 0.5 l Sequenase, 1 l as usual, do this just before starting reactions Termination reaction: Although the starting volume is slightly larger than in the USB protocol, still add 4 l of labelled DNA into 2.5 l of each dITP for termination reactions, add 4 l stop solution. Notes: 1. We can read at least 400 bp for each reaction. 2. This procedure works very well for sequencing double stranded plasmid DNA.