Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Summary

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Abstract

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Introduction

Here, two related protocols are presented for the lysis of Caenorhabditis elegans to make DNA accessible for PCR-based applications. PCR is a commonly used molecular technique used for many applications, including genotyping and amplifying DNA fragments for cloning and sequencing, among others. The small (1 mm), free-living roundworm C. elegans is a popular animal system for biological research. Obtaining suitable genomic DNA from a single animal or a few animals is sufficient to amplify the sequence by PCR. Late L4 larvae and adults contain only ~1,000 somatic cells (including some multi-nuclear, polyploid cells), germ cells, a....

Protocol

1. C. elegans maintenance

NOTE: N2 (wild type) and blmp-1(tm548) C. elegans strains were maintained on standard nematode growth media (NGM) plates at 20 °C.

1. Prepare NGM plates by dissolving 23.005 g of NGM powder in 973 mL of water in a 2 L flask. Cover the flask opening with aluminum foil (or autoclavable cap that allows venting) and secure the covering with autoclave tape. Autoclave to dissolve the powder and sterilize the contents with a sterilization cycle of at least 30 min.
2. Cool the medium to 60 °C in a water bath.
3. To the cooled medium, add 25 mL of sterile 1....

Results

Genomic DNA from a single or a few wild-type adults was extracted using the commercial kit or traditional lysis protocol to compare the efficacy of these two methods. These lysates were then used as templates for PCR to amplify either a larger target of ~2,100 bp (encoding blmp-1) or a smaller target of ~500 bp (encoding a part of sma-10). Both methods successfully yielded appropriate PCR products (Figure 1A).

Next, the ability of kit-extracted g.......

Discussion

Determining the genotypes of C. elegans is an important step while performing genetic crosses to create new C. elegans strains. Genomic DNA extraction using a single or few C. elegans is a crucial step in genotyping C. elegans. This protocol describes genomic DNA extraction from C. elegans using a commercial kit. This method is fast and works robustly. The genomic DNA extracted using this method can be used for downstream applications, including genotyping, sequencing, and clo.......

Materials

Name	Company	Catalog Number	Comments
autoclave tape	Defend	43237-2
aluminium foil, heavy duty	Reynolds Wrap	2182934
calcium chloride	Millipore Sigma	102382 (CAS 10035-04-8)
Extract-N-Amp kit (includes Tissue Preparation Solution, Extraction Solution, and Neutralization Solution)	Sigma-Aldrich Co. LLC	XNAT2-1KT
Isotemp hotplate/stirrer	Fisher Scientific	11-100-495H
LB media, Lennox, capsules	MP Biomedicals, LLC	3002-131
magnesium sulfate, 97% pure, anhydrous	Thermo Scientific	AC413485000 (CAS 7487-88-9)
microcentrifuge	Labnet International, Inc.	PrismR, C2500-R
NEB Q5U Hot Start High-Fidelity DNA polymerase	New England Biolabs, Inc.	M0515S	"Pol E" used in Supplemental Figure S1, a high-speed, high-fidelity polymerase (20–30 s/kb)
NGM media powder	US Biological Life Sciences	N1000
Phusion High-Fidelity PCR Master Mix with HF Buffer	New England Biolabs, Inc.	M0531S	"Pol D" in Figure 1B, a high-speed, high-fidelity polymerase (15–30 s/kb)
primers	Integrated DNA Technologies	custom DNA oligos
PrimeSTAR GXL polymerase	Takara Bio Inc.	R050B	"Pol C" in Figure 1B, a high-fidelity polymerase (1 min/kb) for GC-rich templates and templates up to 30 kb
Quick-Load Purple 2-log DNA Ladder (0.1–10.0 kb)	New England Biolabs, Inc.	N0550S
SapphireAmp Fast PCR Master Mix	Takara Bio Inc.	RR350A	"Pol A" in Figure 1B, polymerase used in Figure 1A, C, D, a high-speed polymerase (10 s/kb) for targets up to 5 kb
Sigma ReadyMix Taq PCR reaction mix	Sigma-Aldrich Co. LLC	P4600	"Pol B" in Figure 1B, a polymerase (1 min/kb) for targets up to 7 kb
SimpliAmp thermal cycler	Applied Biosystems	A24812
stir bar	Fisher Scientific	14-512-126
vortex mixer	Fisher Scientific	2215365
worm pick	Genesee Scientific Corporation	59-AWP