20.109(S14):Begin Western protein analysis (Day2)

Introduction

Protocols

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 later evaluate and purify the DNA using gel electrophoresis.
• To avoid pipetting very small volumes, you will prepare a reaction cocktail that uses no less than 1 μL of any restriction enzyme.
  • 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 for each of the above reactions (digest 1 and digest 2) that includes water, buffer and enzyme. Prepare enough of each cocktail for 4 digests. Leave the cocktails on ice.
2. Aliquot 4 μL of the appropriate plasmids into five well-labeled eppendorf tubes. The labels should include the plasmid name, the enzyme(s) to be added and your team color.
3. Add 21 μL of the appropriate cocktail to each tube. 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 return to Part 3 of the protocol.
4. Before leaving lab today, please add 2.5 μL of loading dye to each of the digests you have assembled. You should also prepare undigested samples of parent IPC and each mutant candidate, containing 4 μL of plasmid, 21 μL of water, and loading dye. We will store the digests and the remaining DNA at –20°C.

For next time

Reagent list

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