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Notes March 1 - Notes March 1 Next tim PCR 65-67 e Feb 22...

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Notes March 1 Next time PCR 65-67 Feb 22- Fig 25-26, pg 917, fig 25.30, fig 25.27; etra figure, fig 25.32; 25.33 Fig 3.24 - If you cut the enzyme with two enzymes, there is a lesser chance that it will reseal - pUC18 o has ampicilin resistance which is a gene that makes proteins that cut up the bacteria o origin of replication (not written on the figure) is necessary and present - lacz gene o encodes B-galactosidase o enzyme that chops the dissacharide lactose glucose + galactose o used usually as a reporter gene o small interruption of the lacz gene by the polylinker is not enough to interfere with the function of b-galactosidase gene; the promoter is upper part of the gene region o junk DNA is also made near the encoding junk amino acids that are not part of natural b- galactosidase o nevertheless you do make the end of the protein, and is a functional protein o however, if you a clone a big piece of DNA and insert it into the polyliner, that interrupts the light purple part of the lacz gene, and you don’t get b-galactosidase epression o no insert makes b-gal o with insert there is a distortion of the protein, and no b-gal is made - polylinker- also called the multiple cloning site contained in the lacz gene o about 30 or 40 nucleotides all in a row with different restriction enzyme sites that are un meaning only 1 in the whole vector plasmid o cloning is done to these restriction sites o if you have a polylinker, you can cut with two different enzymes and drop out the tiny pi between those o if you cut the vector with two different enzymes it reduces the chances of resealing sinc are different and not complementary - X-gal o Lacz gene makes b-galactosidase which can cleave the colorless compound x-gal to a bl product; has been introduced in mice as well o Cleaved similar as lactose is cleaved o no insert makes b-gal, appears blue o with insert there is an insertion of a protein which makes no b-gal, colonies appear color o use indicator dye called x-gal to detect if your expressing b-gal o cells of E. coli that have been transformed by an unmodified pUC18 plasmid form blue
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