Designing a Cloning Experiment: Maps, Gels and Primers | StemPy Lab
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“Designing a Cloning Experiment: Maps, Gels and Primers” is available to Standard subscribers.
Before a student cuts any DNA, a plan is made on paper. You choose the enzymes from a restriction map, so that the gene goes into the vector in the right direction. You read gels and colony plates, with the controls that tell you whether to trust them. You design primers, and you learn what an expression vector must hold. It goes beyond the textbook, to the level of A-Level Biology, IB Higher Level and the Olympiad.
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Choosing enzymes from a restriction map: A cloning experiment is planned on a map. Two different enzymes at the two ends of the insert make it go into the vector in one direction only. The enzymes must not cut inside the gene. A diagnostic digest with an asymmetric site tells which way an insert went. This lesson works all of these with exact fragment sizes.
Reading gels and colony plates: A gel and a plate are read with the help of controls. A ladder turns the distance of a band into a size. The plasmid DNA has three forms that run at different speeds. A plate of the cut vector alone shows the background. The efficiency of competent cells is counted as colonies for each microgram of DNA.
Primers, annealing and expression vectors: A primer is designed with a length of 18 to 25 bases and 40 to 60 per cent G and C. Its melting temperature sets the temperature of annealing. Tails with a restriction site can be added to the 5 prime end. An expression vector needs a promoter, a ribosome binding site, a start codon in the right frame and a stop codon.