Methods: Wet Lab

Wet Lab Methods

The wet lab is a fragile ecosystem that requires precision and care. Before working in the wet lab ensure you have completed all trainings.

For a reminder about sterile technique please see:

Here are the posted Wet Lab Rules:

SAFETY FIRST
Know where the first aid, fire blanket, and emergency eye station are.
No food, drink, or other consumables
Do not work in the wet lab alone (or at least let someone know you are there)
Put on gloves!
When working with dangerous or corrosive reagents wear safety goggles and a lab coat
Label everything you make with your initials and date
Put away anything you are not actively using
Keep your space and the lab organized
Clean your dishes
Do not leave frozen or refrigerated reagents out for extended periods of time.
Please limit crying in the wet lab while others are working
If faced with a fire in the lab: pause, think, remove it's source of oxygen/fuel, and always report. Water is NOT the answer.

Autoclave magenta boxes (10 seeds per box) one flask, and one beaker* (optional but I like to pour my seeds into a beaker, I have a hard time getting forceps into the flasks) per maize variety.
Make Hoagland Agar, mix 1.63g of Hoagland’s (found in the fridge) and 15 g agar / liter of purified H2O. These numbers can be found on the ingredient bottles.
After autoclaving the mixture you can keep it in the water bath at 55 degrees to keep it from solidifying.
Using the serological pipet put 15mL of Hoaglands Agar into each magenta box and allow to cool.
Put maize seeds into flask and label both the foil and the flask with tape.
Put 50mL of bleach, 50mL of purified H2O, and 2-3 drops of tween 20 into the flask. Swirl vigorously and let sit for 5 minutes.
Decant and rinse with purified H2O 5 times. I like to use the foil that the flask was autoclaved in to keep it covered, and to keep the seeds in the flask when you pour out the liquid.
Fill flask with 100 mL of purified H2O. Place in 60 degree water bath for 15 minutes to heat treat the seeds.
Let the seeds soak in the purified water for at least an hour after you pull them out of the bath.
Place ten seeds in a labeled magenta box with solid agar. Space the seeds out the best you can.
Wrap the lid of the box with parafilm to decrease contamination.

Printing Labels is one of the most challenging tasks you will take upon yourself in the Wallace Lab. Here is a guide to limit the pain.

See this guide in the Journal of Visualized Experiments: Aseptic Laboratory Techniques: Plating Methods. (We especially use the Spread-plating technique with a rod)