Granato M et al. (1990) Worm Breeder's Gazette "RAPID SMALL SCALL WORM PREP"

Schnabel R, Schnabel H, Granato M

[Worm Breeder's Gazette, 1990]

Identification of a molecular polymorphism associated with a recombination event is a time consuming experiment because of the large number of DNA preps. The following procedure has been used to isolate high molecular weight DNA which can be restricted from small quantities of worms. The procedure avoids any CsCl centrifugation or the use of phenol and is therefore time saving. The protocol makes it possible to process easily 20 samples within a day. The yield is about 10 g from three 9cm plates. Procedure: Wash the worms ( starved L1 for highest yield) from the plates and purify from bacteria by standard sucrose flotation in a 1. 5ml cup. Wash once with H2O and pellet the worms (about 0.2ml worm pellet). Leave the cup at least 10 minutes on dry ice ( or store until use at -70 C) and add 0.5ml lysis buffer (50mM Tris-HCl,pH 8.5, 100mM NaCl, 50mM EDTA, 1% SDS) and 30 l Proteinase K (10mg/ml) to each cup, invert gently until pellet is thawed and incubated in a 65 C waterbath for 2 hours. Add 30 l Proteinase K, invert several times and incubated for another 2 h at 65 C. Add 200 l 5M KAc to the viscous solution, invert gently and incubated for 30 minutes on ice. Centrifuge 3 minutes at 13k rpm on a bench centrifuge and transfer the supernatant into a fresh cup. Add 2 volume of ethanol by overlaying the ethanol and mix by inverting the cup gently. Leave 20 minutes at RT and wind up the precipitated DNA with a bent glass capillary. Transfer the DNA to a new cup with 200 l TE where it should dissolve easily. Overlay with 200 l isopropanol and mix slowly by inverting the cup. Leave 20 minutes at RT. Again wind up the DNA as before and wash the DNA by placing it with the glass capillary in a cup with 400 l 70% ethanol for 3 minutes. Transfer the DNA as before into a cup with 40 l TE plus 100 g/ml RNase A. Heat for 10 minutes at 65 C to dissolve the DNA and place the solution onto a millipore filter floating on TE in a 3cm petri dish. Remove the dialysed DNA after one hour from the filter and check the concentration and quality on a 0.8% agarose gel. Millipore filters: pore size 0.025 m (VSWP01300), place on TE, shiny side up.

Finney M et al. (1986) Worm Breeder's Gazette "big DNA."

Finney M, Horvitz HR

[Worm Breeder's Gazette, 1986]

We have been using Maynard Olson's one-dimensional field-reversal gels (an improvement on Schwartz-Cantor orthogonal gels; see below) to look at large pieces of DNA. This technique is potentially useful for worm breeders in several ways. First, it may be possible to separate small free duplications from worm chromosomes, making quick clone mapping or region-specific libraries possible. Second, restriction enzymes that cut worm DNA infrequently can be used to determine the structure of DNA in a large region; specifically, they can be used to find breakpoints of chromosomal aberrations. Third, resolution can be improved over normal gels in the 10-50 kb range. Intact (>2Mb) DNA was prepared (see below) from SP957, a strain carrying mnDp30 balanced by a deficiency. On gels that resolve a 1.6 Mb yeast chromosome, mnDp30 is excluded from the gel (using a probe kindly supplied by Barbara Meyer). We were not particularly surprised, as 1.6 MB is about one-eighth of a chromosome, about the genetic size of mnDp30. (Contrary to Olson, we find that DNA larger than a certain cutoff size remains at the origin; he probably did not see this phenomenon because he used nothing larger than yeast chromosomes.) The eight-base recognition sequence enzymes NotI and SfiI both cut worm DNA into a smear of fragments ranging in size from about 50 kb to about 300 kb (and a small amount of DNA of very high molecular weight). This range is somewhat smaller than was predicted based on the AT content of worm DNA. Getting a complete digest can be difficult if there is very much DNA; nevertheless, we do see bands on Southerns, and we are looking for the eT1 breakpoint using probes linked to unc- 86.Technical Section Intact worm DNA: Prepare nuclei suspended in agarose as follows. Freeze worms at -80 C (or in liquid nitrogen) in a buffered solution containing 1mM spermidine, 5mM EDTA, and 1% NP40. Grind the frozen worms in a mortar until powdered. Scrape the powder into a microfuge tube and let thaw on ice. Spin in a microfuge for 1 second to pellet debris and transfer the sup to a new tube. Spin for 10 minutes at 5 C to pellet nuclei and discard sup. Resuspend pellet in the same buffer without detergent and add and mix in quickly an equal volume of 50mM EDTA 1% agarose, melted and cooled to 55 C. Let the agarose harden and slice the plug into convenient sized pieces. Add a ml of your favorite SDS-proteinase K solution and incubate at 65 C for an hour. Soak the slices in several changes of TE. Store them at 5 C in TE. To digest, soak a slice in the appropriate enzyme buffer containing 1mM of the protease inhibitor PMSF, and then change the buffer and add enzyme. Before trying to load the slice into a gel well, it helps to soak it in TE containing dye, so you can find it if you drop it. Field reversal electrophoresis: First read Carle, Frank, and Olson, Science 232, 65, 1986. Initially we reversed the field using a computer-controlled apparatus built by Leon Avery and MF. Plans are available on request. The disadvantage of this arrangement is that the computer is tied up for the entire run of the gel. Now we are using a switching apparatus with an integral microprocessor built by MF. We are still experimenting with conditions--for the latest, give us a call.

Bakaev VV et al. (1998) Worm Breeder's Gazette "AN ATTEMPT TO SLOW AGING IN C. elegans. 9. NO POSITIVE EFFECT OF EPITHALAMIN"

Bakaev VV, Efremov AV, Anisimov VN

[Worm Breeder's Gazette, 1998]

There are data on the life span prolonging effect of pineal peptide preparation epithalamin in mice, rats and Drosophila melanogaster [1,2]. The purpose of this study was to investigate the effect of different concentrations of epithalamin in water solutions on nematode life span. In this experiment epithalamin was used in following dilutions 1:10*5, 1:10*6, 1:10*7, 1:10*8, 1:lO*9 and 1:10*10. Three adult animals (3 - 5 days old) were kept in microtitre wells containing 0.75 ml of liquid medium (with E. coli and without epithalamin) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (with epitalamin in any concentration) every day (one worm in one well). This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Table I Concentration of Longevity (days) epithalamin n mean +/- S.E. maximal Control 12 17.4 +/- 2.5 29 1:10*5 12 12.8 +/- 2.1 27 1:10*6 12 15.1 +/- 1.9 28 1:10*7 12 17.1 +/- 2.2 28 1:10*8 12 19.6 +/- 2.2 29 1:10*9 12 18.8 +/- 2.0 31 1:10*10 12 18.7 +/- 1.7 25

Ishii N et al. (1985) Worm Breeder's Gazette "C elegans oncogenes."

Ishii N, Suzuki K, Tomita S

[Worm Breeder's Gazette, 1985]

The existance of cellular oncogenes in C. elegans was investigated. DNA was isolated by the method of Emmons (Gazette, Vol. 3, # 2, p. 34) from the eggs of C. elegans. About 0.5 g of eggs isolated by treatment with hypochlorite from about 5x10+E4 adults were suspended in 2.5 ml of a solution containing 0.1 M NaCl, 0.1 M EDTA, 0.05 M Tris- HCl (pH 8.0) and were ground to a powder in a mortar on dry-ice. Proteinase K (Sigma) was added to 200 r/ml and the mixture was incubated overnight at 37 C. 300 l of the mixture was suspended in 3. 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and 3.7 g CsCl were added. After centrifugation at 32,000 rpm for 48 hours, the purified DNA was obtained. DNA samples were digested with the restriction enzyme EcoRI and subjected to agarose gel electrophoresis (8%, 20 V, 20 hours) and filter-blot transfer to Zeta-Probe Blotting Membrane (Bio Rad). Filter-blotted DNA was prehybridized(#1) and hybridized(#2) with a nick-translated [alpha-P32]dCTP-labelled DNA probe (K-ras and V-myc genes) under two sets of conditions, nonstringent(#3) and stringent(#4) hybridization. Hybridization was revealed by autoradiography for two days at -70 C (XAR-5 film, Kodak). One K-ras and three or four V-myc oncogenes were identified in C. elegans.1) Incubation was performed in a solution containing 5x SSC, 0.05 M sodium phosphate buffer (pH 6.5), 10x Denhardt's, 0.5 mg/ml salmon sperm DNA, 50% folmamide, 5% Dextran sulfate (M.W. 5,000) for ten minutes at 85 C and overnight at 42 C. 2) Incubation was done in a solution 5x SSC, 0.05 M sodium phosphate buffer (pH 6.5), 2x Denhardt's, 0.2 mg/ml salmon sperm, 50% folmamide, 10% Dextran sulfate for ten minutes at 85 C. 3) Incubation was done in 2x SSC, 0.1% SDS for 60 minutes at 65 C and shaken overnight at room temperature. 4) After the nonstringent hybridization, incubation was done in 0. 1x SSC, 0.5% SDS for 120 minutes at 68 C. After the solution was changed, incubation was continued for a further 60 minutes at 68 C.

Yanase S et al. (1998) Worm Breeder's Gazette "One tube RT-PCR assay from single worms"

Ishii N, Okano M, Yanase S

[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

Gerber A et al. (1990) Worm Breeder's Gazette "Cloning ced-1, a gene required for the engulfment of cell corpses"

Gerber A, Horvitz HR, Ellis RE

[Worm Breeder's Gazette, 1990]

In wild-type C. elegans hermaphrodites, 131 cells undergo programmed cell death. Each resulting cell corpse is quickly engulfed and degraded by a neighboring cell. Ed Hedgecock showed that mutations in the genes ced-1 and ced-2 prevent the engulfment of cell corpses. Since then, we have identified five additional genes -- ced- 5, ced-7, required for the engulfment step. In animals with mutations in any of these genes, corpses resulting from programmed cell death can persist for hours. Genetic studies suggest that these genes fall into two groups that might have partially redundant functions in the engulfment process: ced-1, ced-7 and ced-8 constitute one group, while ced-2, te the other. To understand better how the engulfment process works at the molecular level and why there appear to be two classes of genes involved in this process, we have undertaken a molecular analysis of ced-1.The ced-1(n1506) I mutation was isolated in a mut-2 genetic background, in which a number of C. elegans transposons are active. Southern analysis revealed a novel 2.9 kb EcoRI band in DNA from ced-1(n1506) animals, using the transposon Tc3 as a probe. This polymorphic band is within 0.1 map units of ced-1.We cloned this Tc3 polymorphism from a size- fractionated library made with DNA from ced-1(n1506) animals. This clone, skT1, contains 2.0 kb of non-Tc3 sequence in addition to a 0.9 kb EcoRI end fragment of Tc3. We used parts of the non-Tc3 sequence of skT1 to probe a copy of the YAC 'polytene' filters generously provided by John Sulston and Alan Coulson. Three overlapping YACs, Y5D9, Y53C10 and Y47H9, which form part of a previously unpositioned 700 kb contig, were identified. None of the cosmids that overlap all three of these YACs hybridizes to our probe, so we constructed a phage library from the YAC Y5D9 by ligating size-selected SauIIIA partial digests of Y5D9 into BamHI-digested Lambda Dash arms. We obtained approximately 125,000 recombinant phage. We screened 50,000 of these with the probe derived from skT1 and identified several thousand positive plaques. We are currently analyzing these phage, with the goal of obtaining 30 to 40 kb of overlapping phage covering the region of the Tc3 polymorphism. We plan to use these phage in microinjection experiments to attempt to rescue the ced-1 phenotype.

Browning H et al. (1992) Worm Breeder's Gazette "Transformation Rescue of spe-11"

Strome S, Browning H

[Worm Breeder's Gazette, 1992]

Mutations in the spe-11 gene (for spermatogenesis-defective) result in a paternal-effect embryonic-lethal phenotype. Fertilization of wild-type oocytes by sperm from mutant animals leads to abnormal early embryonic development. This and other studies suggest that the sperm must contain or supply the spe-11 gene product for normal early development to occur (Hill et al. Dev. Biol. 136, 154-166). In order to molecularly characterize the gene, we are cloning spe-11 by transformation rescue using YACs, cosmids and phage. spe-11 maps .1-.2 m.u. Ieft of dpy-5 .We have co-injected a number of the cosmids and YACs from this region (provided by Alan Coulson and John Sulston) with rol-6 plasmid into + spe-11 dpy-51 unc-73 + + heterozygotes and tested homozygous spe-11 dpy-5 progeny for rescue. The YAC Y51G8 rescues both spe-11 and dpy-5 mutant phenotypes. This results in roller animals of wild-type length, which produce Dpy and wt length but no Unc animals. When these wt length animals are crossed to N2 males, all the Fl progeny produce some spe-11 dpy-5 animals. Thus, the genotype of these animals is spe-11 dpy-5 / spe-11 dpy-5 ,with the YAC rescuing both spe-11 and dpy-5 . We hybridized Y51G8 to a Southern blot of some of the cosmids in the region [See Figure 1] and injected those cosmids to which it hybridized. The cosmid B0342 rescues dpy-5 .However, none of the cosmids we tested appear to rescue spe-11 .This could be due to deletions in the cosmid DNA we isolated. We are suspicious that cosmids from this region are prone to rearrangements. In some cases the vector bands are not the expected size, overlap with other cosmids is not as extensive as expected, and different isolates of the same cosmid show different restriction patterns. Consequently, rather than requesting additional cosmids from this region, we chose to screen an unamplified lambda DASH II C. elegans genomic library with the rescuing YAC and Y38H4 .Since by Southern analysis Y38H4 ends in B0342 ,we further characterized 22 phage which hybridize to Y51G8 but not Y38H4 . We established the order of the phage by hybridization to cosmids which hybridize to Y51G8 and began injecting phage from the most suspicious areas. Lo and behold, one such phage with an insert of approximately 18kb rescues spe-11 .This phage lies on the very left edge of Y51G8 (the YAC hybridizes to only part of the phage) between C5 OD8and F55A12 .F55G5 should cover this region; however, our isolate does not We are currently determining which cosmids overlap with the phage. In addition, we are injecting restriction fragments from the phage to zero in on the gene. (The library and phage are available upon request.).

Hope IA et al. (1988) Worm Breeder's Gazette "transformation to the rescue."

Thierry-Mieg D, Hoskins R, Hope IA, Sulston JE, La Volpe A, Spence AM

[Worm Breeder's Gazette, 1988]

In order to make good use of the genome map (see update, this issue), we need an efficient means for searching out loci that have been shown genetically to lie within a limited region. Transformation rescue, developed by A. Fire (EMBO J. 5(10) pp.2673-2680 1986), offers a potentially more powerful, and also more stringent, identification of loci than has previously been available. It is, of course, also central to the identification of functional elements by reverse genetic analysis (eg Fire, WBG 10(1)). Since hearing from Andy Fire that he had been successful in obtaining transformants with DNA minipreps, we have been using a very simple protocol to prepare DNA for injection. Cosmid DNA is extracted from 2ml bacterial culture by a standard alkaline lysis procedure, ethanol precipitated and redissolved in 200 l TE. An equal volume of 4.4M LiCl is added, and the mixture is left on ice for an hour. RNA and other contaminants are pelleted by centrifugation and the DNA is precipitated from the supernatant with two volumes of ethanol. The DNA is reprecipitated from 100mM KOAc (Fire,1986) and dissolved in 10 l of injection solution. The size of the cosmid DNA should always be checked on an agarose gel to guard against large deletions. We use the injection set-up and procedure of Fire (1986). We are following his advice in injecting under paraffin oil rather than Voltalef. We find that it is unnecessary to have Lucifer Yellow in the injection solution as successful injection is easily visible with Nomarski optics. We periodically verify that the needle is flowing by touching it to the agarose pad. It is possible to obtain rescued animals by injecting nuclei or by actively avoiding them, but we do not know what influence the site of injection has on the transmission of the rescuing DNA. We have observed rescue arising in the offspring of unrescued progeny of injected animals. Whenever possible, when searching for a gene, we inject a set of cosmids which overlap to the extent that each region is represented in two cosmids. This should control against the presence of small rearrangements in any given cosmid and increase the chance of having the entire gene on at least one cosmid. Our experience is admittedly limited, and some mutants may well prove difficult to rescue, but we have not yet encountered a gene refractory to transformation or a DNA clone which is known to span a gene and yet fails to rescue mutants in that gene. It is worth pointing out that unc-32 and unc-30 are, as far as we know, the first cases of nematode genes being cloned on the basis of genetics, physical linkage, and rescue, without the involvement of polymorphisms in mutants. As the physical map becomes more crowded, this approach to cloning loci can be expected to become increasingly general and convenient. [See Figure 1]

Morton DG et al. (1986) Worm Breeder's Gazette "screens for maternal-effect lethal mutations affecting the gut lineage."

Morton DG, Sprunger SA, Kemphues KJ

[Worm Breeder's Gazette, 1986]

In an effort to understand the mechanism of determination in early C. elegans development, we have been using a screen devised by J. Priess et al. (C. elegans newsletter 8, #2, 5) to identify maternal- effect lethal (mel) mutations that affect the early embryonic cleavage pattern. This procedure requires first identifying the mel mutants, maintaining them in stock by selection, then observing early cleavages in embryos from mutant homozygotes of each strain. This approach has resulted in the identification of several mutants with aberrant cytoplasmic partitioning, defining three loci, designated par-1,2,3 ( Kemphues and Priess, Abstracts, 1985 C. elegans meeting, p15). However, because of the low frequency of par mutants among the total pool of maternal effect lethals, observing early development is not an efficient way to obtain large numbers of mutants with this phenotype. In addition, there may be mel mutants that affect determination without detectably affecting cytoplasmic partitioning. In an effort to overcome these problems we have begun to screen for mels that produce embryos arresting with large numbers of cells but which do not differentiate gut cells. This can be done very simply by scoring newly identified mel homozygotes (which are bags of dead embryos) under polarized light to visualize gut granules. Gut granules normally appear in cells of the gut lineage at about the 200 cell stage of embryogenesis. Mutants that produce embryos with large numbers of cells but no visible gut granules are potentially defective in some step in the determination or differentiation of the gut. In this screen, those mels that produce embryos with gut granules are discarded as are mels producing embryos with no gut granules and obviously small numbers of cells. We expect to find at least three interesting classes of mutations in such screens: 1) New alleles of par-1. Mutations in par-1 (b274, e2012) have been shown to affect partitioning of cytoplasm in the early cleavages and lead to arrested embryos with many differentiated cells, but no detectable gut granules. 2) Mutations in other loci that affect partitioning of cytoplasm. Because cytoplasmic localization appears to be important for gut determination, multiple loci controlling localization could mutate to a similar terminal phenotype. 3) Mutations that specifically affect the determination or differentiation of the gut. If there are maternally expressed molecules with a unique function in the determination or differentiation of the gut, mutations in genes encoding these molecules should surface in our screens. Our initial results are promising. We have screened roughly 3000 F1 clones from mutagenized adults and have identified four mel mutants whose progeny arrest in embryogenesis with large cell numbers but no gut granules. One of the mutants (it32) is a new allele of par -1 Two other mutations identify a new locus par-4 (it33, it47) V (near dpy-21). Progeny from par-4 mutants are defective in P granule localization and have synchronous early cell divisions, defects common to all par mutants. However, par-4 mutants differ from mutants at the other par loci in that the first division results in nearly normal size asymmetry. The fourth mutant has no obvious partitioning defect, but early divisions are slowed about two-fold compared to wild-type. Relative timing of divisions is normal, and the E lineage is normal at least to the 8 E-cell stage. The low frequency at which these mutants arise, coupled with the relative ease of screening should make it possible for us to satura te the genome for loci that can mutate to this phenotype.

Plasterk RHA et al. (1993) Worm Breeder's Gazette "Isolation of Tc1 Insertion Mutants From the Mutant Bank: An Update"

de Vroomen M, Korswagen HC, Plasterk RHA

[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.