Talk:20.109(F09): Journal Club I

From OpenWetWare

Jump to: navigation, search

Contents

Introduction

To visualize the amount of β-galactosidase associated with the colonies transformed with the library, we will overlay the indicator known as Methylumbelliferyl-β-D-galactoside, aka "MUG." This histochemical yeilds methylumbelliferone, a fluorescent molecule, when it is cleaved by β-galactosidase. Moreover, it's freely diffusable into live cells, thus can be applied to petri dishes for direct observation of the transformed cells under UV light. That is what we'll do today.

Protocol

Before the Journal Club Presentations

  1. Mix 100 ul of MUG with 2.5 ml molten top agar
  2. Pour over surface of plate
  3. Allow to solidify and react at room temp.

After the Journal Club Presentations

  1. Observe colonies under a hand-held long wavelength UV lamp held a few inches from the surface of the plate.
  2. Choose two colonies that are either more bright than the others or more dim--depending on the phenotype you're looking for.
  3. Circle the colonies on the back of the plate and give the plate to the teaching faculty. If the colonies are crowded you may be asked to restreak the colonies and will be shown how.
  4. Colonies will be grown in liquid culture for your next day of lab.


Reagents

  • 10 mg/ml MUG in DMSO
  • Top Agar
  • Hand held UV lamps
Personal tools