Simply Cloning - Chapter 3 - Vector Restriction Digest

Simply Cloning is a video manual for making DNA constructs.
Chapter 3 is a bench demonstration of vector restriction digest.
Narration Script:
During the vector restriction digest we are going to incubate our plasmid with a pair of restriction enzymes that will cut plasmid DNA and will produce a linear DNA fragment with sticky ends. The protocol for setting up a vector restriction digest is shown here and in the Protocols poster.
Vector restriction digest protocol:
1. Prepare restriction mix
o 39 µl ddH2O
o 5 µl 10X enzyme buffer
o 5 µl DNA (approximately 1 µg)
o 0.5 µl restriction enzyme A (5 units)
o 0.5 µl restriction enzyme B (5 units)
2. Mix up by pipetting up and down
3. Incubate at 37 oC for 45 min
For the vector restriction digest I prepared distilled water, NEB buffer 3, pSAT6, which is the plasmid I am going to cut, an empty Eppendorf tube and the enzymes that I am going to use, which are XhoI and BamHI.
In the empty Eppendorf tube I am going to combine:
• 39 µl of water
• 5 µl of NEB buffer 3
• 5 µl of pSAT6 plasmid from a minprep
• Half a microliter of XhoI
• And half a microliter of BamHI
I will mix it up by pipetting, label the tube, and put it in a 37 oC incubator for 45 min.

[@user-ci7fe1jj8x]

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[@CloningStrategies]

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[@khoapham7120]

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[@123range27]

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