C. Elegans Unit: Lab Activities

Overview of Techniques

Basic Worm Handling Techniques www.silencinggenomes.org

Pouring NGM Agar plates Nematode Growth medium

Innoculating broth with OP50 E. coli for feeding

“Seeding” NGM Agar plates with OP50

Chunking worms to propagate more worms

Picking worms to move exact numbers of worms Making a worm pick

Chemotaxis Assay – Biorad Kit

Basic Technique: Pouring NGM Plates Melt down 1-2 bottles of NGM agar

Hot plate, water bath, microwave

About 14 plates per group of 2

Label with Initials

Date

NGM agar

INSTRUCTOR NOTE: HAVE a student pour 16 extra plates for instructor use with Chemotaxis kit (worm thawing)

Basic Technique:Innoculating with

OP50 Using sterile technique, transfer 3mls of LB

broth (premade) to a clean bacterial culture tube (the clear ones with loose caps)

Dip a sterile innoculating loop into the OP50 stock broth or touch to surface of stock plate culture

Put loop into your LB broth and swirl

Label with your initials, date, and OP50

Put in tube racks in 37°incubator

NOTE: All materials that touch bacteria go in bacterial waste pans

Basics: Seeding NGM plates with OP50

Add OP50 in red sharpie to the labels on your plates

Once OP50 has grown for at least 24 hours, Add 200 μl of OP50 broth culture to the surface of 6 of your NGM plates (2 for chunking, 2 for picking, 2 for making worm stocks for chemotaxis assay later) Leave any remaining plates empty for later –store in

fridge

Using an L-spreader, spread the broth around, avoiding the very edges of the dish.

Let soak in about 15 min, then parafilm

Invert and incubate at ROOM TEMP.

NOTE: All materials that touch bacteria go in bacterial waste pans

Basic Technique: Chunking worms

Obtain a stock plate of N2 (wild-type) worms from instructor

Get 2 of your NGM + OP50 plates, Add to the labels “N2”

Flame-sterilize a scalpel, and touch to surface of agar to cool

Cut a 1cm2 square of agar from the stock plate

Transfer it to the surface of the NGM-OP50 plate

Parafilm and invert

Incubate at room temperature

Basic Technique: Picking Worms

Instructor will demonstrate how to make a worm pick

Make your worm pick and label it with your initials

Get a plate of worms that was chunked a few days ago and has many worms on it

Transfer 5-10 worms to an NGM + OP50 plate

Practice!

Video: https://www.youtube.com/watch?v=eTQtkQm5hOw&feature=related

Biorad Chemotaxis Assay: Quick Guide

Pour NGM agar plates, Seed with OP50 To make stocks of N2 and Mutant worms for

assay

Use Wash buffer (S-buffer) method to transfer worms to make a new stock plate

Pour assay plates - 20 total per kit

Prepare Assay plates

Perform assay

Collect & record data

Perform Statistical analysis

Biorad Kit

Prepare NGM seeded with OP50 You have extra from our basic worm

techniques – you’ll need just 2 plates for this assay

You should have 2 plates left with NGM & OP50, no worms – if not seed more.

NOTE: the kit calls for use of OP50-pBAD which has an amp resistance gene, and requires culture on NGM+amp. We are skipping this as it is not necessary and gives us more flexibility.

Biorad Kit: Pour assay plates

These have a small amount of Wash buffer in the agar instead of NaCl (a component of NGM)

We have just 20 per class (per kit)

Instructor has prepped solution & autoclaved it; Each team come pour 2 plates

Label with your initials, date and ASSAY plates

Biorad Kit: Making stocks of WT and Mut

(daf-18) worms Use wash buffer transfer method (S-buffer

technique) to make new stocks of your worms onto NGM + OP50 (NOT ASSAY PLATES)

Follow instructions on pp. 30-31 of your kit manual Make sure you label plates with WT worms

(N2) or Mutant (daf-18)

Don’t cross contaminate them

Be sure to incubate at room temperature

Need 5 days to make enough worms for next steps

Sec.

BioRad Kit: Finishing assay plates – adding

salt gradient Instructor has prepared wash buffer and

2.5M NaCl solution

Follow instructions on next slide to set up plates

Let plates incubate overnight before use.

BioRad Kit: Chemotaxis Assay

Follow the instructions on pp. 35 -38 of your handout to set up chemotaxis assay

Allow 30 minutes for worms to move

Collect data & record in notebook

Next class: Perform Statistical analysis

Chi Square Analysis

Follow along with BioRad kit Chi-Square supplement

Read it

Try practice problems

Analyze your own data

Record in notebook