Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Protokol

The following protocol is an excerpt from Prior et al, A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins, J. Vis. Exp. (2018).

Single Worm PCR and Genotyping

1. After 1 - 2 days of egg-laying, transfer the F₁s into individual PCR strip tube caps containing 7 µL of Worm Lysis Buffer using a worm pick (Table 1, Figure 1D, day 4 - 5).
2. Centrifuge PCR tubes at maximum speed for 1 min at room temperature to bring animals to the tube bottom, and freeze tubes at -80 °C for 1 h.
   NOTE: Worms can be stored at -80 °C indefinitely.
3. Lyse frozen worms in a thermocycler using the following program: 60 °C for 60 min, 95 °C for 15 min, 4 °C hold.
4. Set-up PCR mastermix as shown in Table 2.
5. Add 21 µL of PCR mastermix into clean PCR tubes, and then add 4 µL of worm lysis from step 3. Mix well using a pipette and run PCR program following the manufacturers guidelines (see Materials Table).
6. Purify PCR reactions using a DNA Clean and Concentrate kit, following the manufacturer instructions (see Materials Table). Elute DNA by adding 10 µL water to the spin column.
7. Set-up restriction enzyme mastermix as shown in Table 3.
8. Add 10 µL of the restriction enzyme mastermix to each cleaned PCR reaction and incubate for 1 - 2 h at 37 °C.
9. Separate digested PCR products on a 1.5% agarose gel run at 120 V using 1x Tris-acetate-EDTA (TAE) buffer.

Sonuçlar

Figure 1. CRISPR-Cas9 engineering of the C. eleganssod-1 locus. (A) Schematic illustration depicting the exon-intron structure of the sod-1 locus in C. elegans. The red bar denotes the location of G93 in exon 3. The primer pair set is noted by the red arrows (F1-R1). (B) S...

Malzemeler

Name	Company	Catalog Number	Comments
Nuclease-free water	Synthego Inc.	provided with the sgRNA kit EZ kit
KCl	Sigma	P5405
DNA Clean & Concentrator	Zymo Research	D4004
Zymoclean Gel DNA Recovery Kit	Zymo Research	D4002
Q5 Hot Start High-Fidelity 2X Master Mix	New England Biolabs	M0494L
Proteinase K	Sigma	P2308
MgCl2	Sigma	M2393
NP-40	Sigma	74385
Tween-20	Fisher Scientific	BP337-100
RNaseZap Decontamination Solution	Fisher Scientific	AM9780