Difference between revisions of "20.109(S14):Complete Western and prepare damaged DNA (Day4)"
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==Protocols== | ==Protocols== | ||
+ | |||
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+ | ===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. | ||
+ | |||
+ | <center> | ||
+ | {| border="1" | ||
+ | | | ||
+ | !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 _____ | ||
+ | |- | ||
+ | | H<sub>2</sub>O | ||
+ | |colspan="4"| For a total volume of 25 μL | ||
+ | |} | ||
+ | </center> | ||
+ | |||
+ | #Prepare a reaction cocktail that includes water, buffer and enzyme. | ||
+ | #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. | ||
+ | #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. | ||
+ | #Before leaving lab today, please add 5 μL of loading dye to your digest. We will store these at –20°C. | ||
==For next time== | ==For next time== |
Revision as of 19:23, 3 March 2014
Contents
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 |
- Prepare a reaction cocktail that includes water, buffer and enzyme.
- 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.
- 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.
- 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
write something here or not accessible to edit
Next Day: Cell preparation for DNA repair assays
Previous Day: Begin Western protein analysis