Lecture4mcb160Lf11

Lecture4mcb160Lf11 - RogerL.Miesfeld2000 Alternate methods...

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Roger L. Miesfeld © 2000
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Alternate methods to CaCl2 Heat-shock transformation: 1. Use DMSO (Dimethyl Sulfoxide) to permeabilize cell membranes.Incubate treated cells with DNA on ice, followed by heat-shock at 42 deg C. 2. Electroporation: Use electric pulses to permeabilize cell membranes. Incubate cells with DNA in low ionic strength medium (e.g. pure water) and apply electric fields.
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Roger L. Miesfeld © 2000 Transformation by Electroporation
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www.inovio.com/images/IMG_electropora>on.gif
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Author: Richard Wheeler
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Anima>ons on transforma>on can be viewed at these sites Heat‐shock: hFp://www.dnalc.org/ddnalc/resources/transforma>on2.html Electropora>on: hFp://www.learnerstv.com/anima>on/anima>on.php?ani=167&cat=biology
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In above drawing, Ligase “seals” a nick in double stranded DNA (e.g. Nicks in DNA replication, from Okazaki fragments)
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EXAMPLE
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EcoRI EcoRI XhoI XhoI Can be inserted in this direc>on Cannot be inserted in this direc>on Directional cloning allows the insert to be ligated in only one orientation. Cleavage of the vector with two different
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This note was uploaded on 01/25/2012 for the course MCB 160L160L taught by Professor Venkatesansundaresan during the Fall '11 term at UC Davis.

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Lecture4mcb160Lf11 - RogerL.Miesfeld2000 Alternate methods...

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