Brood Size Assay

This assay has been performed with both Cry5B crystal spore lysates from Bt cultures (22) as well as with Cry14A expressed inside E. coli (23). However, in principle this assay should work with any purified Cry toxin or any soluble toxin. 1. Attach an artificial eyelash to a wooden pick by dipping the tip of eyelash in fingernail polish and then applying eyelash to end of pick. Trim eyelash so approx 3 mm extends from end of pick. Prepare a batch of such picks, wrap in aluminum foil and...

Dual Excitation Ratio Imaging

Although many types of microscopes will work, our preference is an inverted differential interference contrast microscope with dual camera ports and a long working distance condenser. The objectives should be of the best fluorescence quality affordable and capable of imaging through both a cover slip and agarose pad (0.17 mm each). Although we routinely use a x20 dual-emmersion objective (NA 1.3) for obtaining images of the intestine, we also use a x60 long working distance dry objective with a...

Measuring Synaptic Currents at the Neuromuscular Junction

Endogenous synaptic currents can be recorded from C. elegans muscles with relative ease because the neuromuscular synapses are typically not disrupted by the dissection. Currents mediated by specific receptor subtypes can be studied in isolation of other currents by using available mutations in genes encoding ion channel subunits, pharmacological antagonists, and by manipulating extracellular or intracellular ion composition. Under typical recording conditions, C. elegans neuromuscular synapses...

The C elegans Neuromuscular Junction A Model Polyinnervated Synapse

Elegans Body Wall Muscle

In C. elegans, specialized projections from the body wall muscle, called muscle arms, extend to the ventral nerve cord where they make en passant synapses with cholinergic and GABAergic neuronal processes. Patch-clamp recordings from C. elegans muscles have revealed a variety of ligand- and voltage-gated currents, including acetylcholine ACh - and GABA-gated ion-otropic receptors as well as voltage-gated K and Ca2 currents 20,29,30 . Elec-trophysiological analysis of mutants with disrupted...

PHSensitive GFP Expression Plasmids

Most GFP variants are sensitive to pH, although with different spectral characteristics and pKas. This sensitivity results from a conformational change following protonation of the chromophore and can be influenced by amino acid substitutions that modulate the conformational switching or that influence protonation through electrostatic interactions. In our laboratory, we have mainly employed pHluorin 12 as a pH sensor, owing to the fact that movement of the worm can be a confounding factor when...

Robby M Weimer

High-pressure freezing HPF is capable of converting liquid water, to a depth of approx 0.6 mm, into amorphous ice nearly instantaneously. At midbody, an adult Caenorhabditis elegans hermaphrodite approaches its widest girth of approx 0.1 mm. In theory, an entire living adult animal can be physically immobilized instantly in amorphous ice by HPF, thus, providing a unique opportunity to examine cellular architecture with exquisite spatial preservation. The following chapter will discuss, in...

Wide Field Fluorescence Microscopy

Imaging of fluorescent probes at a wide-field microscope i.e., your standard fluorescent microscope is straightforward with modern microscopes. A few recommendations for optimal image acquisition follow 1. Choice of microscope you do not need the latest, computerized model for viewing and documentation of still images. In the author's lab, people have the choice of a 12-yr-old Axioskop and a fancier, motorized Axioplan II same manufacturer . Everybody wants to use the old Axioskop, because...

DNA Templates for dsRNA Production

For cloning gene fragments into plasmid-based templates choose a vector with the appropriate bacteriophage promoter sites Fig. 1, Constructs 1-4,6 and appropriately placed restriction sites. Plasmid vectors are available from several suppliers of molecular biology products. pBlueScript Stratagene has T3 and T7 promoter sites that could be used for Constructs 1, 3-6. A double T7 plasmid L4440, generated by the Fire Lab 3 , Fig. 1, Construct 2 and Fig. 2 is used in the feeding protocol and is...

Vectors Containing cDNAs for Fluorescent Proteins

The Fire lab vector kits are the major source for GFP vectors for use in C. elegans www.ciwemb.edu pages resources.html . The kits are now distributed by Addgene http www.addgene.org Andrew_Fire . The vector kits contain vectors with cDNAs of all relevant GFP variants. The following variants are recommended GFP in this text refers to the S65C variant named gf3 in the 1999 Fire kit . CFP corresponds to the Y66W N146I M153T V163A variant gf15 and YFP is the S65G V68A S72A T203Y variant gf42 ....

Programmatic Access to GFF Databases Using BioDBGFF

To retrieve genomic annotations including anything that may have sequence coordinates , use the Bio DB GFF Perl API part of the BioPerl project 19 . The Bio DB GFF module provides methods for creating and querying relational databases of genomic annotations. These databases are created from tab-delimited files of genomic annotations in the GFF format. Files contain one annotation per line with fields describing its reference sequence typically a chromosome , its source how the annotation was...

L1 Growth Assay

Earthworm Anterior And Near Med

On day 1, inoculate 5 mL of Luria-Bertani LB medium with a single E. coli OP50 colony and incubate overnight at 37 C and 250 rpm. Also bleach gravid worms on d 1. 2. The assay is set up on day 2 using a 24-well plate. Each well will contain 40 L of E. coli OP50 in S medium, 5 L of L1 worms in M9 medium 30-40 worms total , 4 L 100X toxin or control buffer, 1.2 L chloramphenicol, and 350 L S medium to bring total volume to 400 L. 3. Specifically on day 2, pellet the E. coli 0P50 culture at...

C elegans Strains and Growth Conditions

The precise conditions for maintaining and growing C. elegans strains will depend on the selection strategy you choose Table 1 . Most strains can be maintained at 20 C on 60 mm nematode growth medium NGM plates seeded with OP50 bacteria 32 . However, strains carrying temperature-sensitive mutations will need to be grown at the appropriate temperature. For growing large quantities of worms for bombardment, use liquid cultures, 100-mm NGM, opti-gro, enriched peptone, or egg plates completely...

Handling of Bacteria From the C elegans RNAi Feeding Library

Gene knockdown via dsRNA mediated interference, or RNAi, is extraordinarily efficient in C. elegans. To generate this knockdown effect, worms can simply be fed bacteria designed to produce dsRNA homologous to a single predicted gene in the C. elegans genome 9,10 . In these bacteria, an ampicillin- Fig. 1. Schematic representation of the genotype of double-stranded RNA dsRNA producing bacteria. Strain HT115 DE3 contains an isopropyl-P-D-thiogalactopyra-noside IPTG -inducible T7 RNA polymerase...

Growing C elegans Strains for Transformation

The following protocols are for preparing large quantities of C. elegans prior to microparticle bombardment transformation. Specific conditions will vary depending on the selection strategy and strain you are transforming Table 1 . Because each of the microparticle bombardment transformation protocols described in this chapter requires a specific number of worms for a transformation, instructions for each protocol are included. 1. Create a master plate of the worm strain you have chosen by...

Tools for Comparative Genomics

WormBase provides several useful tools and precomputed datasets for comparative genomics analyses. First, WormBase houses the entire C. elegans and C. briggsae genomic sequences and predicted gene and protein sets. Second, pre-calculated reciprocal best mutual match BLASTP orthologs between C. briggsae and C. elegans are displayed on the Gene Summary page when known . Finally, WormBase displays nucleotide level alignments between C. elegans and C. briggsae on both the Genome Browser and the...

PCR Detection of Deletions

The mutant libraries contain 4608 sample populations. Rather than testing all of these by PCR individually, we strategically pool the samples in a way that balances the requirements for the sensitivity of the assay with our desire to identify the particular source of the positive sample with a minimal number of PCRs. In one pooling strategy, we collect and pool the samples from one microtiter plate into a single sample. The initial screening PCRs, done in duplicate on the pooled samples, lead...

Programmatically Mining the Web Interface

A first step toward programmatic data mining at WormBase is to create scripts that extract data directly from pages on the website. This approach is often referred to as screen-scraping. Screen-scraping scripts can be created with limited knowledge of a programming language such as Perl. Although not an official WormBase data mining method, the ease with which these scripts can be created make them a viable strategy for fetching data from the site. Screen-scraping offers several advantages over...