Nematode Growth, Mutagenesis and Harvest
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| Growth Medium: For 1L of rich NGM (RNGM) |
| 3g NaCl |
| 7.5 g Bacto Peptone |
| 15g Agarose |
| Autoclave and cool to 55o |
| Add: |
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1ml cholesterol (5mg/ml in ethanol, flammable!)
1ml 1M CaCl2
1ml 1M MgSO4
25ml 1M KH2PO4, pH 6.0
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| Notes: The peptone is 3x standard NGM. We use agarose to inhibit the burrowing of worms. Not all brands of agarose work for this purpose. We recommend Electrophoresis Grade Seakem LE agarose cat number 50004 from Cambrex. Biotechnology Grade agarose from Amresco does not work for this purpose. |
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| Growth and Synchronization for Mutagenesis Figure 1 |
| We grow worms on 5-10 15cm RNGM plates to generate a large population of gravid adults.
We wash the gravid adults off of the plates and treat with basic hypochlorite to harvest the eggs. Briefly, we collect worms by centrifugation in M9 Buffer (22mM KH2PO4, 22mM Na2HPO4, 85mM NaCl, 1mM MgSO4, add 10 volumes of basic hypochlorite (0.25M KOH, 1-1.5% hypochlorite, freshly mixed), and incubate at room temperature for about 4 minutes. Eggs (and some residue of carcass) are collected by centrifugation (400g, 5 minutes, 4o). We wash the eggs 5x with 10 volumes M9 and collect by centrifugation. We distribute the eggs across 10-20 15 cm plates made with RNGM and seeded with E.coli OP50.
After 52 hours of growth at 20o the resulting worms are collected and mutagenized.
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| Mutagenesis |
| We have recovered deletions after mutagenesis with three different mutagens: UV/TMP, EMS and DEB. Protocols for their use are summarized by Anderson (1996). Briefly, after synchronization (Figure 1) the worms are washed off the plates with M9 Buffer. We then split the population three ways and mutagenize. |
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| UV-TMP - Our procedure was modified from Yandell et al. (1994). To an 8ml suspension of worms in M9 Buffer we add 8ml of M9 containing 4,5',8-trimethylpsoralen (Sigma #T6137)Z at 60 mg/ml (prepared using a 3mg/ml TMP stock in DMSO). We incubate the worm suspension in a horizontal, capped 50ml centrifuge tube at room temperature in the dark for fifteen minutes. We then transfer the worms in the dark to a sterile, glass 15cm petri plate and irradiate the suspension with 360 nm UV light for 90 seconds at 340 mW/cm with gentle shaking. To assemble the UV source we mount two hand held UV lamps (Model UVL-21 Blak-Ray Lamp, longwave UV-365 nm, Fisher Scientific , #11-984-40) using appropriate clamps on a ring stand. We adjust the height of the lamps to generate the appropriate UV dose as measured with a UV dose meter (Model UVX Digital Radiometer with UVX-36, Fisher Scientific #97-0015-01). |
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| EMS - To an 8ml suspension of worms in M9 Buffer we add 8 ml of M9 containing 100 mM methane sulfonic acid ethyl ester (Sigma #M-0880). We incubate the worm suspension in a horizontal, capped 50ml centrifuge tube at room temperature for four hours with moderate shaking. We then wash the worms 5x with 10 volumes of M9 Buffer. After each wash the worms are collected by centrifugation at 1000g at 4o for 5 minutes. |
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DEB - To an 8 ml suspension of worms in M9 Buffer we add 8 ml of M9 containing 0.2 mM 1,2:3,4-diepoxybutane (Sigma #D7019). We incubate the worm suspension in a horizontal, capped 50 ml centrifuge tube at room temperature for three hours with moderate shaking. We then wash the worms 5x with 10 volumes of M9 Buffer. After each wash the worms are collected by centrifugation at 1000g at 4o for 5 minutes.
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| The mutagenized worms are plated on a total of 10-20 15cm RNGM plates seeded with OP50 and allowed to grow for 24 hours. (TMP treated worms are kept in the dark). We then collect F1 eggs by treating the worms with basic hypochlorite as described above (Figure 1). We allow the eggs to hatch on the surface of a 15cm RNGM plate without OP50. We then plate the resulting F1/L1 larvae. |
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| Library Plating |
| We collect the F1/L1 worms in M9 Buffer. After careful counting we make a suspension of worms with a concentration of 500 worms/ 0.1ml. We then distribute the worms in groups of 500 onto 1152 6cm OP50 seeded RNGM plates using a repeating pipettor with sterilized syringes. We store the plates in groups of 96, (labeled A-L), in lidded plastic containers. We allow the worms to grow for five days at 20o-21o. |
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| Library Harvest |
| We number the plates in groups of 96 and place them in stacks of six (Figure 2). Using a repeat pipettor we add 0.75ml of sterile distilled water containing streptomycin (100 mg/ml) and Mycostatin (12.5mg/ml) to each plate in a single stack. We rock the plates to dislodge the worms and, using a standard micropipettor, we transfer 150ml of the worm suspension from each of the six plates to the appropriate six wells of a deep, 96-well microtitre plate (DyNA Block 1000 deep well microplates, Midwest Scientific, #P9615). To keep track of the well position, as we harvest we temporarily eject the spent micropipette tip into the well. |
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| DNA preparation |
| Once we fill a 96 well microtitre block with worms we add 150ml of Proteinase K solution to each well (50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween-20, 0.01% Gelatin, 200mg/ml Proteinase K) (Figure 2). We then seal the blocks with a mat lid (Flexible mat lid, 96 well, Midwest Scientific, #P9618). We freeze the worms at -80o for 20-30 minutes and then incubate the blocks, with intermittent mixing, for six hours to overnight at 65o. We find that the lids do not seal the wells completely so we seal the perimeter with tape prior to a prolonged incubation and we are careful to avoid vigorous mixing that may lead to well-to-well contamination. |
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| Sample pooling |
| At this stage a portion of the worms from each of the 1152 plates are distributed into twelve deep 1ml 96-well microtitre blocks (Figure7). Each sample represents the DNA from 500 F1 animals or 1000 mutagenized genome equivalents. Prior to pooling, we dilute each sample with an equal volume of sterile distilled water. We pool the samples in a two dimensional array; we pool both the rows and columns from each of the twelve plates such that the twelve initial plates are reduced to two sets of 96 samples. Eight samples in the pooled rows, and twelve samples in the pooled columns, for example, represent the 96 samples from set A. To make the pools we transfer 50ml of a given sample (in sets of eight or twelve) to the appropriate wells of a deep 96-well microtitre plate. We centrifuge the pooled samples to pellet the cuticle residue that remains after the Proteinase K treatment. Approximately 150ml of each cleared sample is transferred to PCR tubes where they are heated to 95o for twenty minutes to inactivate the Proteinase K. For the pooled columns we use PCR tubes in strips of twelve (FisherBrand 12 Strip 0.2 ml thin-wall reaction tubes, #05-407-3A with caps, #05-407-4B. For the rows we use strips of eight tubes (MJ Research 8 Strip 0.2 ml reaction tubes, TBS-022, with caps, TCS-0801). |
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| PCR |
| We do nested PCR (Figure 3). In general, we select primer sets that are approximately 3-3.5 KBp apart, though we have had success with primers as close as 1KBp. We select primers that are about twenty bases long with about a 50% G/C content. |
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| The PCR reactions are done in 96-well PCR trays (MJ Research 96-well PCR tray, #MPL-9601) sealed with dimpled rubber mats (Perkin Elmer 96-well plate cover, #N801-0550). |
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| First Round PCR |
| Each reaction is 25ml and contains the following: |
| 5ml Template DNA |
| 10 pMoles of each outside primer |
| 2.5ml of a 10x dNTP stock (10x=2mM) |
| 2.5ml of a 10x Assay Buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 25 mM MgCl2) |
| 0.5 units of Taq polymerase (Fisher Biotech - FB6000-55) |
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| The reactions are done in a thermocycler from either MJ Research (Figure 4) or Biometra Figure 6. We use the following cycling conditions: |
| 94°, 30 seconds, 1 cycle |
| 92°, 30 seconds; 55o, 20 seconds; 72o, 2 minutes; 35 cycles |
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| Second Round PCR |
| Each reaction is 25ml and contains the following: |
| 10 pMoles of each inside primer |
| 2.5ml of a 10x dNTP stock (10x=2mM) |
| 2.5ml of a 10x Assay Buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 25 mM MgCl2) |
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| The template DNA for the second round reaction is transferred from the first round reaction using a 96-pin replicator (Boekel Replicator, Fisher #05-450-9). The replicator transfers an insignificant volume to the reaction and so the reaction mix must be adjusted appropriately. If you insist on using a micropipette to transfer the first round to mix to the second round reaction, you must dilute the first round reaction 5-10 fold. |
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| Finally, we have tested reaction volumes as low as 10ml for the second round PCR with reasonable success. |
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| Identifying Candidate Populations and Sib Selection |
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| Gel Electrophoresis |
| Our initial screen is done on 96 samples representing the pooled rows. We examine the PCR reaction products on gels using a gel system configured for the analysis of samples in microtitre arrays (Figure 5). Samples that show bands that are shorter than the wild type are selected for further analysis. We substantiate the validity of the deletion band and determine the precise address of the candidate sample by repeating the PCR on the twelve appropriate samples from the pooled columns. For example, if a candidate is determined to originate from group A, we retest only those pooled columns from group A. It therefore takes a total of 108 PCR reactions to identify the address of a candidate population. If a band appears in the appropriate samples after PCR of both the rows and columns, the candidate is validated and we proceed to the sib selections. |
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| Sib Selection |
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| First Round |
| We wash a portion of the worms off the sample plate with M9 buffer. We count the worms to determine the concentration, dilute appropriately, and distribute the worms in sets of 50 into the wells of 12-well tissue culture plates (Evergreen Scientific, #222-8046-01F) containing RNGM and seeded with OP50. In the first round we set up 192 populations. We grow the worms for five days and harvest a portion of each population for PCR. To harvest we flood the well with 200ml of sterile distilled water containing streptomycin and Mycostatin as described above, and transfer 50ml of the worm suspension to a deep well microtitre plate. It is convenient to use a repeating pipettor to fill all twelve wells in a single plate with water, tip the plate to a 45o angle, and extract the worm suspension using a standard micropipettor. In this way, a practiced technician can harvest 192 samples in twenty minutes. We add an equal volume of Proteinase K solution to the 50ml worm suspension, freeze the samples at -80o, and incubate at 65o for four hours. We then add an equal volume of sterile distilled water to each sample and centrifuge to pellet the cuticle residue. |
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| We transfer 5ml of each sample to a 96-well PCR tray using a 12 channel pipettor. We heat the tray to 95o for fifteen minutes to inactivate the Proteinase K. We then add 20ml of appropriately prepared PCR reaction mix to each sample and repeat the nested PCR as describe above. |
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| Second Round |
| Based on the data, we collect worms from a candidate population, as above, and distribute in sets of 10 into the wells of 24-well tissue culture plates (Evergreen Scientific, #222-8044-01F) containing RNGM and seeded with OP50. We set up 96 populations. We grow the worms for five days and harvest as above except that we flood the wells with 100ml of sterile distilled water and transfer 25ml to a deep 96-well plate. The Proteinase K and PCR conditions are as described above. |
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| Third Round |
| Based on the data, we collect worms from a candidate population, as above, and transfer a portion to several 6cm RNGM plates seeded with OP50. After one to two days we pick single worms from these plates into the wells of 24-well tissue culture plates containing RNGM and seeded with OP50. We set up 192 clones. We harvest a portion of each population after four to five days and test with PCR as above. |
| Figure1 |
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| Figure4 |
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| The following text was copied from the MJ Research web site: |
| The PTC-100 has been a laboratory stalwart since 1989. |
| Improved Hot Bonnet heated lid |
| Six standard block styles |
| Famous MJ Research user-friendly software |
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| Figure5 |
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