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[
Worm Breeder's Gazette,
1980]
If you are peacefully picking worms one day and an eight-legged hairy monster walks into the field of your dissecting microscope, after you come down from the ceiling, you will know that you too have a mite infestation. You will also know the plot for a grade B horror flick. A piece of the moth crystals used in closets (paradichlorobenzene) placed in your incubator for a few weeks will kill them off with no detectable harm to worm stocks.
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[
Worm Breeder's Gazette,
1989]
The map is now widely distributed electronically (see WBG 10(3), 67), but we are once again providing a summary for the gazette in the form of an output from the routine CHPLT. Do note that this is a provisional best guess, and that some linkages may later go away: please enquire if you need to know about the status of particular areas. When you receive cosmid clones, as stabs, please IMMEDIATELY streak them out on selective medium, pick small colonies, and grow 4ml minipreps (protocol from Alan Coulson if needed). For some cosmids, larger preps are liable to yield deleted DNA. Check that cosmid DNA appears full size (runs slower than lambda on agarose gels), then freeze a sample of good cells in 20% glycerol at -70 C. MRC computer account 'ARC' does not exist; Alan and John share account JES. A database node is now open at Seattle: modem number 206-467-2957; operator Phil Meneely. The summary of clone types given on the next page may be helpful when you are deciding which clones to request for your research. To reveal the most suitable clones for microinjection, the buried clones need to be displayed by the routine CONTASS; we will help you to do this if you ask. [See Figures 1- 3]
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[
Worm Breeder's Gazette,
1984]
In the last few months, several people have asked about the CGC strain freezing procedures. For general reference, here are some detailed notes and observations on freezing and thawing for optimum survival. Two important things. The most important thing is that you have lots of L1's and L2's to freeze, as they are the best survivors. L3's and L4's seem to survive at a lower rate, and adults do not survive well at all. The other important thing is that freshly starved worms seem to survive freezing better than well-fed ones. Getting lots of L1's and L2's to freeze. Homozygous stocks are generally pretty easy. If you start a large agar plate (15 x 90 mm, streaked with a grid of OP50) with ten L4 hermaphrodites, the plate will starve when the F2's have recently hatched. This is a very happy thing, and you can proceed to the actual freezing. With heterozygous stocks, you'll need to start a few clones on small (15 x 60 mm) plates, check the progeny for correct marker segregation, and pick three confirmed clones. When these starve, wash off all three plates and pool the worms for freezing. Other types of stocks are a little more tedious. These include burrowers, male stocks, paralyzed or severe Dpy and other slow-growers. With any of these types, it sometimes helps to start three or four small plates with one to five L4's, or three or four cross plates in the case of male stocks. As soon as you get a bunch of L1's and L2's on the plates, wash them off thoroughly, pool and wash the worms free of bacteria by centrifugation and removal of the supernatant. Resuspend the worms in 2 ml of M9 buffer and put them on a shaker at an appropriate temperature for 12 hours or so. Overnight is fine. This starves them. Before freezing, add one drop of an overnight OP50 culture so the worms will have plenty to eat when thawed, and freeze as usual. This isn't as much work as it sounds, and gives you a warm feeling later. It is especially useful with burrowers (most wild-type C. elegans isolates), of which there are usually none left on the surface when the plate starves. Actual freezing. Having obtained lots of larvae in a starved state, this is what I do: (1) Wash larvae off the plate with 2.2 ml of M9 buffer, draw the buffer/worm suspension into the pipette, measure ( usually 1.6 to 1.8 ml) and place the suspension in a sterile 30-40 ml test tube with closure; (2) add an equal volume of S medium plus glycerol (this is 30% glycerol, w/v, so the final is 15%); (3) vortex gently to mix; (4) draw up 3 ml in a sterile 5 ml pipette; and (5) dispense in 0.6 ml aliquots to five 2 ml Nunc freezer vials. These vials are placed in holes drilled into a styrofoam block which has been fitted into a styrofoam box that has a lid (Lew and Miwa, WBG 4: #1), and the whole thing is put in a -80 C freezer. Leave them there at least three hours; I usually leave them overnight. Then you can put them in liquid nitrogen. Four of the vials go into twin LN2 refrigerators; the fifth is put in liquid nitrogen in a separate place to be thawed the next day as a survival tester. Survival and contamination checking is done on the third day after thawing. If you have three small plates instead of one large, start with 2.5 ml of buffer to allow for what soaks into the agar. Wash one plate and draw the mixture back up, wash the next two plates with that. If you're doing the shaker trick, just add an equal volume of S + glycerol, vortex and dispense. It helps to use disposable graduated conical tubes with screw caps, since you can easily see how much S + glycerol to use. You can also prepare a single freezing solution, a 50/50 mixture of M9 buffer and S + glycerol, and use that to wash off the plates. You can either start washing with enough solution to leave 3 ml for dispensing into vials, or wash with a smaller volume and bring it up to 3 ml just before dispensing. Thawing. In my experience this is not terribly critical, but I try to do it the same way always. Vials are removed from liquid nitrogen and placed on the lab bench, and a timer is set for ten minutes. When the timer goes off, you'll usually have a chunk of ice with a layer of liquid around it in the vial. If it's still frozen solid, rub it briskly in your hands until the pellet starts to melt. Wipe the vial dry with a Kimwipe. This keeps condensed water from the outside of the vial from falling onto the petri plate. Vortex with the cap still on to mix the stuff into a runny slush and dump it into a clean, labeled agar plate. Results. At the beginning of the first Genetics Center contract, N2 freezing survival testers were frozen to provide monthly and yearly tests over the entire contract period. Survival rates from those testers averaged 26% with no decrease in survival in stocks frozen for the entire 5-year period; fluctuations around that average are presumably due to sampling variations. Long-term survival tests will continue. Survival of stocks stored at -80 C in a Revco is as good as in liquid nitrogen over an initial 6-month test period.
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[
Worm Breeder's Gazette,
1993]
As some of you are already aware, Academic Press has asked Henry Epstein and myself to edit a volume of Methods in Cell Biology devoted to C. elegans. One of the useful appendices which we would like to include is a list of available antibodies and protocols for using them. If you have generated an antibody in your laboratory which you would like to see listed in a methods volume on C. elegans, please contact me as soon as possible. Incidentally, if you would like to make your antibodies widely available but are nervous about being swamped with requests, you might considered contributing your monoclonal antibodies to the Developmental Studies Hybridoma Bank sponsored by the National Institute of Child Health and Human Development. They are providing the wonderful service of growing up monoclonals and making the cells or supernatants available to researchers at reduced fees. I know from personal experience that their anti-tubulin antibody works well for C. elegans, and it is considerably cheaper than commercial products. Contact Dr. Thomas August, Johns Hopkins University at (410) 955-3985 for more information about receiving a catalog or making contributions. I have the impression that they are not anxious to handle hundreds of worm specific antibodies, but that they are interested in handling a limited set of the most widely used C. elegans specific antibodies as well as antibodies which are known to cross-react with other species.
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[
Worm Breeder's Gazette,
1995]
As many of you know, there is a gopher server at the CGC(elegans.cbs.umn.edu) and a World-Wide-Web (WWW) server in my lab inDallas
(http://eatworms.swmed.edu/) devoted to information about Celegans. These servers are heavily used.There are several other C elegans information servers, but they are notincluded here because I don't have their usage information.The table shows that, although many connections come from net-surfers whovisit once and never again (perhaps having expired from boredom), there isa large group of repeat offenders who use the servers over and over. Many, perhaps most, come from C elegans labs. (For instance,wormworld.ucsf.edu and horvitzlab3.mit.edu are pretty obvious.)My point is, if you need to reach the C elegans community, this is a goodway to do it. There is an Announcements page on both gopher and WWW. Inthe 55-day period analyzed it was read 203 times. If you have a jobopening or a conference or anything else you'd like worm people to knowabout, you probably should be advertising there. Announcements can besubmitted by e-mail to leon@eatworms.swmed.edu, and usually appear withina few hours.
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[
Worm Breeder's Gazette,
1993]
Most of the points covered in the accompanying 'Nomenclature Guidelines -- New Recommendations' contribution were circulated by e-mail before the June 1993 meeting, to principal investigators with e-mail addresses, for their comments and reactions. This procedure appeared to be efficient and helpful. We wish to expand our e-mailing list for nomenclature issues, and for recommended methods of data submission. For this reason, we sent a message in August 1993 inviting other potentially interested parties to join this list, using the e-mail addresses from the 1993 WBG Subscriber Directory. We repeat this message below, for the benefit of readers who have e-mail access but did not receive our invitation. If you are interested, send us an e-mail message asking to be added to the list. From cgc@mrc-lmb.cam.ac.uk Dear WBG subscriber At present, CGC e-mail messages about methods of data submission, nomenclatorial issues and so on, are sent to all investigators with assigned lab codes (strain and allele designations) and known e-mail addresses. We would like to extend this mailing list to other interested parties who can be reached by e-mail. If you wish to be included on this expanded e-mailing list, please reply to this message, and you will be added to the list. Investigators already on the list need take no action. Regardless of whether you join the list, you will still be able to take note of any significant new recommendations, because these will be duly announced in issues of the Worm Yours sincerely, Jonathan Hodgkin Mary O'Callaghan
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[
Worm Breeder's Gazette,
1997]
We are preparing a supplementary vector kit for release February 1 1997. If you are interested in obtaining these kits or documentation concerning their use, please email us (fire@mail1.ciwemb.edu), and we will return the relevant release forms. The 1997 supplementary kit contains the following goodies:
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[
Worm Breeder's Gazette,
1992]
After tabulating the results of the Worm Plate Survey. we have come up with some interesting results. Most notably. the high variability in prices that labs are paying for their plates, even for the exact same plates from the same supplier, and the fact that most plates are marked up considerably over the actual cost. The replies can be separated into 4 categories: Labs that get plates from Fisher ($29-$58). but wish they had non-vented plates Labs that get non-vented plates via Applied Scientific (~$38) Labs that get plates from Falcon (vented) or Nunc (non-vented) and pay much more Most labs' plates were "slipable" or "semi-stackable", but all labs wanted plates that stack well for easy manual pouring, seeding, carrying, and using. Everyone wanted plates with shallow lids such that the bottoms can be lifted out of the tops for inverted use. Some labs expressed an interest in plates slightly smaller than "60 mm". That number is in quotes because all of the companies' plates have bottoms smaller than 60 mm (e.g. Fisher -54 x 14 mm). We have negotiated with the plastic companies that really make the plates for Fisher, Applied Scientific, etc. (that actually just resell them to you). I have come to the conclusion that we can provide you with better worm plates, the same worm plates cheaper, or in most cases better worm plates cheaper. This is true for every lab. The bottom line is that we can get you top quality non-vented "60 mm" plates (like Applied Scientific's, except fully stackable) for about $29 per 500 case INCLUDING shipping depending on your usage and how many cases you can receive at one time. Several labs have found the non-vented plates last longer without drying out or getting contaminated, compared with normal vented plates, so you should save that way, too. We offer full service shipping (e.g. standing orders and same-day telephone orders, free. Similarly low prices are available on 100 mm and 150 mm plates that exceed industry standards for flatness (reducing media usage) and clarity. The 100 mm are about $27 per 500 case plus shipping; The 150 mm dishes (good for DNA & RNA preps and library platings, with more than 2.25x the surface area of 100 mm dishes) are made thicker and deeper than industry standards and are about $21.50 per 100 case plus shipping. The shipping charge is very low for labs, or groups of labs in one city, that can take delivery of many cases in a single shipment. You can even suggest that your stockroom order plates from us. Call us for an exact price quote depending on your usage and how many cases you can receive at one time. In any case, we'll work things out to save you money. In the future, we can offer inexpensive 35 mm dishes if the community at large can order about 2000 cases per year, so let me know about your needs for other sizes. The response was very mixed about pre-poured plates. We may set that up later, but for now we can help the most by saving you lots on empty petri dishes (and later, maybe media .supplies). We are happy to send out free samples so you can examine the dishes. If we haven't contacted you yet, just give us a call. Respondents: 38 (including 5 anonymous) "Winners": Horvitz = 550, Meyer = 400, Thomas = 400, Greenwald = 300 200-299 cases 8 labs 100-199 cases 7 labs 4-99 cases 19 labs Highest price per case: US = 118.75, Canada = $117 (non-vented) Lowest price per case: US = $29, Canada = $25 (vented) Farthest away response: Malta! No responses from MRC or anyone else in Europe or Asia. It is possible that we can save money and/or provide better plates for these labs, including, shipping, too. Let us know.
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[
Worm Breeder's Gazette,
1993]
After the worm meeting 1993 we have received several requests to isolate Tc1 insertion alleles from the frozen library. Marianne de Vroomen has joined the lab, and spends most of her time on the isolation of mutants from the library. This abstract gives an overview of the genes that we have looked at or are looking at. Thus far insertion alleles of most genes can be found; nevertheless we are still expanding the library and improving the technique (see Korswagen et al., Meeting 1993, p. 254). We have obtained one or more lines with insertions in the following genes:
gpa-1 pgp-1 prk-2 pes-9 gpa-2 pgp-2 ceh-6 lin-26 gpa-3 pgp-3 ceh-13 flp-1 gsa-1 pgp-4 ceh-18 ZK637 .5
goa-1 prk-1 ceh-22 gpb-1 odc-1 We have (partial) addresses for the following genes (which means that usually -but not always- a clonal line can be obtained): dynamine
crf-2 ncc-1 ZK637 globin gene GluR40 PCTAIRE-kinase
cap-1 SRp30b
gqa-1 We have not yet looked through the complete library at the following genes: SOD (II)
ubc-1 lir-1 pes-10 SOD(III) SRp20
kin-15 and
kin-16 pes-1 elt-1 apl-1 We have failed to find an insert and will try with new primers for the genes:
ubc-2 cmk-1 cm14 h10 wnt-1 We are still ready to look for insertions in your favourite gene(s). If you want us to do that, please send an E-mail and send primers that follow the guidelines below. The closer you stick to the guidelines the more searches we can do. One point may need clarification: having only a cDNA sequence one could in theory also design primers, and test them on genomic DNA to check that they do not cross an intron-exon boundary and that the prime sites for the two nested oligonucleotides map close to each other. But it is much better to have at least a few kbp of genomic sequence: that way you are 100% sure of the primer sites, you can later choose primers at the appropriate distance to select deletion derivatives, and you can determine the precise insertion site of Tc1 even when it has integrated into an intron. Guidelines for primer choice and shipping To do a search for a Tc1 allele of a sequenced gene we would like to have: 1. a print of 3 kbp of continuous genomic sequence, with the exons of the gene indicated, and the primers indicated (with their direction!). If you have a bit less than 3kbp of sequence information we can do a search nevertheless; the 3kbp is based on our experience that is an ideal distance to choose primers to select deletion derivatives of a Tc1 allele. 2. Two sets of nested primers, each at one end of the 3 kbp, and pointing inward. We like them as follows: -two primers pointing the same direction, not further apart than necessary (100, 200 bp is fine). -approx 50% GC, 21-23 nt long, no funny sequences (palindromes, repeats or identical sequences), somewhat balanced composition of all 4 nt, and at the 3' end a G or C. -Primers in TE, concentration set at 100 M (100 l is plenty). You can send them without dry ice or anything, but perhaps it is good to use Federal Express or an equivalent 24 hour service. -Life is easy for us if you use a simple name for the primers. E.g. the first three letters of your name, plus a number. So e.g. JON I, JON2 ,JON3 and JON4 .Then make sure that JON2 is the nested primer of JON I, and JON4 the nested primer of JON3 . Please mark the tubes on the side and on the top, and make sure the label or marking sticks. In principle one rather than two sets of primers is sufficient, but it gives us an extra option, and you can later use the primers to select deletion derivatives. Also, having two sets of primers at this distance allows you to do a PCR with all 4 combinations to check that the primers are in order (this is not necessary if you know that your oligo-synthesis can normally be trusted). If you ask for insertions in more than one gene, please indicate your priority.
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[
Worm Breeder's Gazette,
1993]
It would be useful if worm labs had access to each other's strain lists. For instance, one lab may have put away in the freezer as uninteresting a mutation that is just what a second lab wants. Unfortunately, there have always been practical difficulties to sharing strain lists: they change frequently, and there is no common format. A new network program called Gopher (see contribution from Mike Cherry on previous page of this WBG issue, which discusses the use of Gopher in connection with accessing the ACEDB database) may be the solution to these problems. Gopher sends an inquiry over the Internet directly to the source, without the user having to know about any messy details. The reply is current, and comes back as text, so that format problems don't arise. Other kinds of information (e.g., pictures) can also be made available. To test the feasibility of Gopher for data sharing, we have set up Gopher service at the CGC (elegans.cbs.umn.edu; IP address 134.84.210.1) and the Avery lab (eatworms.swmed utexas.edu; IP address 129.112.11.21). By gopher to either of these addresses you can get the CGC bibliography, strain list, WBG subscriber directory, recent WBG tables of contents, Avery lab strain lists, pictures of mutants, manuscripts in press, and (thanks to Mike Cherry at Massachusetts General Hospital) access to ACEDB information. Gopher is available for Macintosh, IBM-PC, Unix, and Xwindows by anonymous ftp from boombox.micro.umn.edu (134.84.132.2). If you don't know how to get it, send e-mail to leon@eatworms.swmed.utexas.edu and we will try to help. Also, we would like to urge other labs to make data available by Gopher. If you do, send us e-mail so you can be included in the CGC menu. If you would like to make your strain lists available but don't know how, send e-mail and we'll see if we can help with that.