20.109(S14):Complete Western and prepare damaged DNA (Day4)
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Revision as of 01:44, 19 March 2014 by AgiStachowiak (Talk)
Contents
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.
- 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.
- Flick the tubes to mix the contents, touch-spin, then incubate the mixtures at 37°C for at least one hour. Write down your start time, in case you get distracted later on.
- 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
Next Day: Cell preparation for DNA repair assays
Previous Day: Choose system conditions and paper discussion