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| | ==Protocols== | | ==Protocols== |
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| − | ===Part 1: Digest plasmid for NHEJ assay===
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| − | *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.
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| − | *To avoid pipetting very small volumes, you will prepare a reaction cocktail that uses no less than 1 μL of any restriction enzyme.
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| − | **Note that enzyme stock concentrations can be found on the NEB product page for that enzyme.
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| − | *The table below shows sample calculations for '''one''' reaction. You will likely need to double or quadruple the volumes below.
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| − | <center>
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| − | {| border="1"
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| − | |
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| − | !Example (1x)
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| − | !Your Digest (1x)
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| − | !Your Digest (scaled up)
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| − | |-
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| − | | Plasmid DNA
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| − | | X μL = Y μg
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| − | | X μL = Y μg
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| − | | N/A
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| − | |-
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| − | | 10X NEB buffer
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| − | | 2.5 μL of buffer CutSmart
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| − | | 2.5 μL of buffer _________
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| − | | ____ μL of buffer _________
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| − | |-
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| − | | Enzyme 1
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| − | | 2.5 U = 0.125 μL of ''ScaI''
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| − | | 2.5 U = __ μL of _____
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| − | | ___ U = __ μL of _____
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| − | |-
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| − | | (Enzyme 2)
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| − | | None
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| − | | 2.5 U = __ μL of _____
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| − | | ___ U = __ μL of _____
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| − | |-
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| − | | H<sub>2</sub>O
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| − | |colspan="4"| For a total volume of 25 μL
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| − | |}
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| − | </center>
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| − | #Prepare a reaction cocktail that includes water, buffer and enzyme.
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| − | #Combine (25-X) μL of the cocktail with (X) μL of of plasmid DNA in a well-labeled eppendorf tubes.
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| − | #*The label should include the enzyme(s) to be added and your team color.
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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.
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| − | #*While your samples are digesting, you can complete the other parts of today's protocol.
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| − | #Before leaving lab today, please add 5 μL of loading dye to your digest. We will store these at –20°C.
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| | ==For next time== | | ==For next time== |
