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

Introduction

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.
• The table below shows sample calculations for one reaction. You will likely need to double or quadruple the volumes below.

	Example (1x)	Your Digest (1x)	Your Digest (scaled up)
Plasmid DNA	X μL = Y μg	X μL = Y μg	N/A
10X NEB buffer	2.5 μL of buffer CutSmart	2.5 μL of buffer _________	____ μL of buffer _________
Enzyme 1	2.5 U = 0.125 μL of ScaI	2.5 U = __ μL of _____	___ U = __ μL of _____
(Enzyme 2)	None	2.5 U = __ μL of _____	___ U = __ μL of _____
H2O	For a total volume of 25 μL

1. Prepare a reaction cocktail that includes water, buffer and enzyme.
2. Combine (25-X) μL of the cocktail with (X) μL of of plasmid DNA in a well-labeled eppendorf tubes.
   • The label should include the enzyme(s) to be added and your team color.
3. 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 complete the other parts of today's protocol.
4. Before leaving lab today, please add 5 μL of loading dye to your digest. We will store these at –20°C.

For next time

Reagent list

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