Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc‐93 gene using PCR

Abstract

PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the nematode Caenorhabditis elegans and use PCR to detect deletions in the C. elegans unc-93 gene. In advance of the exercises, wild-type and three different unc-93 deletion mutant strains are grown, harvested, and frozen by the instructor. In one approach, students isolate genomic DNA from each strain using a genomic DNA isolation kit and use agarose gel electrophoresis to analyze the DNA and to estimate its concentration. PCRs using primers directed to two different regions of the unc-93 gene are carried out on the genomic DNA from wild-type and mutant strains, and the PCR products are analyzed by agarose gel electrophoresis. Students analyze the gel to determine the approximate location and size of deletions in the three mutant strains. Alternatively, students may lyse single nematodes and carry out PCR in one laboratory session. These exercises should be easily adaptable to detection of well characterized deletions in any organism.

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

“…After 96 h a single female adult nematode was dissected out from G. mellonella cadaver using a platinum needle and placed into a PCR tube with 20 lL lysis buffer (50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl 2 , 0.45% Nonidet P-40, 0.45% Tween 20, 0.01% [w/v] gelatin and 60 lg/mL proteinase K). The mixture was incubated at À80°C for 15 min and then incubated at 60°C for 1 h and 95°C for 15 min in a BIO-RAD DNA Engine Ò thermocycler (Lissemore et al, 2005). The resulting DNA samples were examined by agarose gel electrophoresis.…”

PCR cloning of partial nbs sequences from grape (Vitis aestivalis Michx)

“…In comparison with screening cDNA or genomic library, PCR cloning is a simple alternative. With its diverse applications and straightforward procedure, PCR has been integrated into undergraduate laboratory exercises to teach students the basic technique [10] and to show them its uses in detecting specific DNA sequences [11][12][13][14] and polymorphisms [15][16][17][18], studying gene expression [19,20], blood typing [21], restriction enzyme mapping [22], sex determination [23], and cDNA and noncoding RNA cloning from yeast [24] and fungi [25], respectively. Here, we report a 4-week laboratory exercise on PCR cloning of partial nbs sequences corresponding to a region between the P-loop and GLPL motifs of potential plant R gene products from North American grape, Vitis aestivalis Michx.…”

Studying Human Disease Genes inCaenorhabditis elegans: A Molecular Genetics Laboratory Project

“…Most groups successfully performed nested PCR (a sample student gel is shown in Figure 1B). Successful use of PCR to detect deletions in the C. elegans genome was also shown by Lissemore et al (2005) in an intermediate-level undergraduate molecular biology course.…”