Difference between revisions of "20.109(S14):Complete Western and prepare damaged DNA (Day4)"
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*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). | *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. | **Note that enzyme stock concentrations can be found on the NEB product page for that enzyme. | ||
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− | + | #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. | |
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− | #* | + | |
#Flick the tubes to mix the contents, touch-spin, then incubate the mixtures at 37°C for at least one hour. | #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 | + | #*While your samples are digesting, you can finish the Western. (Or at least start finishing it!) |
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+ | ===Part 2: Complete Western protein assay=== | ||
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+ | ===Part 3: Gel purify digested plasmid=== | ||
==For next time== | ==For next time== |
Revision as of 01:39, 19 March 2014
Contents
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.
- 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.
- 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