Simply Cloning - Chapter 3 - Vector Restriction Digest скачать в хорошем качестве

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.