Transfer the P0 worms on Plate 2 to a new plate (Plate 3). more precise for 12-channel pipettors in order that they can both seal well when the tips are put on, and also release well a test sample to monitor the viability of the frozen animals. Place the flask on a 20°C shaker overnight (or longer). (where RNAi of one gene inhibits the intended target gene as well as a closely related gene) using BlastN to compare the sequence )X35, 72°X5 min., 4° hold. time. (2003). Examine the phenotypes of F1 worms on Plate 1. rrf-3 and eri-1 both display sterility at 25°C and smaller broods than N2 at lower temperatures (Simmer et al., 2002; Kennedy et al., 2004), but this can be overcome by mating with N2 males after injection, as they are cross fertile at all temperatures (J. Ahringer, The an empty, sterile 15 ml plastic petri dish (not filled with agar) and let sit on the lab bench for 4–5 hours. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. to get through the process. out to test for knockdown of each gene. Two days after the matings were set up, genotype the hermaphrodite mothers (by duplex PCR). As many live worms as possible must be recovered from the thaw. Reagent reservoirs, sterile (Pierce, 15075), 96-well microtiter dishes (Corning/Costar, C3595) or (BD Biosciences, 353072), Microtiter plate brayer (Diversified Biotech, BRAY-4000), “Sidewall tough-tags” labels for the sides of microtiter dishes (Diversified Biotech, TT-SWALL), 100X Penicillin/streptomycin (Invitrogen, 15140-122), 100X Nystatin suspension 10,000 units/ml (Sigma, N 1638), U-bottom 96-well MicroTest III Flexible Assay Plate (BD Biosciences, 353911), Film to seal plates of live worm cultures for freezing, Or MicroAmp Clear Adhesive Films (Applied Biosystems, 4306311), Styrofoam box for freezing live worms (New England Biolabs, small enzyme shipping containers), pH 6.7 1:1 phenol:chloroform mix (American Bioanalytical, AB11051), Thermal cycler (MJ Research, PTC-100HB-96AgV), Microtiter format polycarbonate V-Bottom microtiter 96-well PCR plates (USA/Scientific, 2796-3330), Microseal A film for PCR (MJ Research, MSA-5001), Taq polymerase, 500 Units (Invitrogen, 10342-020), dNTP mixture (dATP, dCTP, dGTP, dTTP), 20 mM each (Amersham Biosciences, 27-2094-02), OWL Centipede extra wide minigel system (Owl Scientific, model D3-14), (this holds two 50 well combs in 2X microtiter spacing), Qiaquick Gel Extraction Kit (Qiagen, 28704), Qiaquick PCR Purification Kit (Qiagen, 28104). bulbs in these machines are for short-wave UV). Nature 408, 325–330. To select primer sets, follow a simple set of rules: 1) primers should be 22mers; 2) they should have 11 Thus, these methods differ from those template: Use T3 and T7 primers to PCR amplify a gene specific fragment cloned into a Bluescript or similar vector that has these primer indefinitely and can be screened at least 400 times. This is a modification of the protocol developed by Ravi Kamath (Kamath et al., 2001), with modifications from Gino Poulin, David Rivers, and Shane Woods. Then, after all the batches nucleotide identity over 200bp, cross-interference is likely. Put plates in a humid chamber at the desired temperature with no shaking. to clean up the genetic background. schedule distributes the work over a three-week period in a fairly even fashion. or worms may die due to lack of oxygen. will be on embryos, then it may not be a good idea to use L1s. the progeny assayed. This part of the protocol will differ slightly depending on your assay. Purchased from Pharmacia as a mixture of dATP, dCTP, dGTP, and dTTP at 20 mM each. If fewer than 100 live worms survive the test thaw then the set If the assay will be on the RNAi treated worms, then starved L1s or later stages can be used. mutant DNA is present at only 1 part in 4,000 in the pooled DNA sample, so the challenge is to increase the abundance of the Add 5 mls alkaline bleach solution to each tube. Try to obtain >1500 per well. the hedgehog in clean dH20, dip in EtOH, flame to get rid of the EtOH, dip in the first round reactions, and then dip in the second round PCR premixes

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