20.109(S14):Complete Western and prepare damaged DNA (Day4)

Introduction

More coming Wednesday. Thanks for your patience!

Brief reminder of where we are. Then…

Topic 1: gel purification and perhaps idea of diagnostic digestion more broadly

Topic 2: western secondary considerations and visualization options

Protocols

Today you get to experience grad student life, juggling multiple assays blah blah blah...

Part 1: Digest plasmid for NHEJ assay

• You will digest pMax-BFP-MCS at the cut site(s) that you chose to investigate last time. We will then evaluate and purify the DNA using gel electrophoresis.
• To avoid pipetting very small volumes, you will either prepare a reaction cocktail that uses no less than 1 μL of any restriction enzyme, or you will prepare an intermediate dilution of said enzyme(s).
  • Note that enzyme stock concentrations can be found on the NEB product page for that enzyme.

1. By whichever approach outlined above, combine 3.5 μg of DNA with water, buffer, and enzyme in a well-labeled eppendorf tube. Whether you prepare an enzyme dilution or a master mix, the enzyme should be added last.
   • Why? What would happen if you added the enzyme directly to water?
   • Recall that you are using 2.5U of each enzyme per μg of DNA.
2. Flick the tubes to mix the contents, touch-spin, then incubate the mixtures at 37°C for at least one hour.
   • While your samples are digesting, you can finish the Western. (Or at least start finishing it!)

Part 2: Complete Western protein assay

Part 3: Gel purify digested plasmid

For next time

Reagent list

write something here or not accessible to edit

Navigation Links

Next Day: Cell preparation for DNA repair assays Previous Day: Choose system conditions and paper discussion