DNA Cloning

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Page 12 Biol 312 L Molecular Biology Lab Blue-white Screening Having the MCS located within the �-peptide gives us a handy screen for plasmids that contain a DNA insert. A solution of “X-gal” (5-bromo-4-chloro-3indolyl-b-D-galactopyranoside) is spread on the petri plate. A plasmid that does NOT have DNA inserted into the �-peptide will produce a BLUE color. However if the �-peptide is inactivated by a DNA insert then the colony will be the normal beige of E coli. We want the boring beige colonies, because they will hopefully have our DNA insert. Figure 3.6 (left) shows the blue colonies of the blue-white screen. The molecular mechanism for blue/white screening is based on a genetic engineering of the lac operon in the Escherichia coli laboratory strain serving as a host cell, combined with subunit complementation from the cloning vector. The vector encodes the � subunit of LacZ protein with an internal multiple cloning site (MCS), while the chromosome of the host strain encodes the remaining � subunit to form a functional �-galactosidase enzyme. The MCS can be cleaved by different restriction enzymes so that the foreign DNA can be inserted within the lacZ� gene, thus disrupting the production of functional �galactosidase The chemical required for this screen is X-gal, a colorless modified galactose sugar that is metabolized by �galactosidase to form an insoluble product that is bright blue, and thus functions as an indicator. Figure 3.7, The blue-white screen. http://en.wikipedia.org/wiki/Blue_white_screen IMPORTANT POINT: This plasmid lets us see the difference between a selection and a screen, two important genetics concepts. The presence of the Ampicillin resistance gene in the plasmid gives us a selection for all bacteria with a plasmid, because they will be resistant to the Ampicillin on the petri plate. By spreading the X-gal on the plate, we can screen for white colonies among the blue ones. These white colonies are our best chance of finding a plasmid with the DNA insert, but it is by no means a guarantee. 2. DNA Insert We will be inserting a fragment of Titin. Titin, which consists of 34,350 amino acids, is the largest known protein. The molecular weight of the mature protein is
Page 13 Biol 312 L Molecular Biology Lab approximately 2,993,442.763 Da, Titin consists primarily of a linear array of two types of protein domains: type I (fibronectin type III domain) and type II (immunoglobulin domain). We are cloning a (roughly) 500 bp segment of the Immunoglobulin domain called Ig27. 3. Cloning the DNA into the Plasmid The Titin DNA is present in another plasmid. Your instructor has used Polymerase Chain Reaction to amplify the Ig27 gene so we can clone it into pGEM3zf. The vector will be cut with BamHI, while the insert will be cut with BglII. Return to page 4 and draw out the pallindromes for BamHI and BglII in the space below. Mark arrows to show where the enzyme cuts the top and bottom strands of the BamHI pallindrome. Examine the BglII restriction site. Where should the BglII enzyme cut so the BglII overhang will match the BamHI overhang? See Figure 3.2 for an example. Can you show the BamHI and BglII cut ends joining together? If so, draw it out below.
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DNA Cloning

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