3.27.07 - 3.27.2007 Needed some other molecule of DNA for...

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3.27.2007 Needed some other molecule of DNA for which we could integrate the human gene we want to clone so that once it is in bacteria it will be heritable Whatever DNA we put it in to make the fragment heritable is called a VECTOR, the one we are talking about right now is a plasmid Step one of molecular cloning o Isolate the human gene you want to clone (won’t talk about it right now because it is the hardest step), must get the particular gene out of its setting, we come back to this at the end o Also you probably wouldn’t do this the first few times you do molecular cloning you’d be presented with the gene already isolated Step 2 o Take the vector, plasmid in this case, and cut it open with a restriction endonucleases Which restriction endonucleases would we use? We want to cut at the lacZ’ gene so we must use one of the restriction endonucleases that cut within the lacZ’ gene, there are many different sites in the multiple cloning site (the point of this site is to provide this flexibility) Step 3 o Put the human gene into this spot, by cutting the gene we have isolated with the same restriction enzyme so it has matching sticky ends, but you don’t want to interrupt the sequence that your trying to clone, we are just cutting the ends of it Step 4 o Then we are going to mix them together and to get ligation we need to add ligase to seal them together Step 5 o Point of molecular cloning is so bacteria can make an unlimited number of copies with a plasmid with this insert so we have to get the plasmid into bacteria o Basically we need transformation: bacteria to take up naked DNA from its environment o Mix the plasmid with bacteria that is competent and add heat to open pores in the plasma membrane and allow the plasmid to slip inside Step 6 o Now ideally you have a tube with an aqueous solution filled with bacteria all that contains the plasmid and all the plasmids contain the human gene we are trying to clone but transformation is not that efficient so most of the bacteria in that tube are untransformed and didn’t take up the plasmid at all o Of the bacteria that did take up the plasmid, most of those took up plasmid that lack the insert This is because of the inefficiency of the ligation step The plasmid has sticky ends to itself so mixing the plasmid with the insert we want to clone, most of the time the plasmid seals itself up with its own sticky ends – this is a NONRECOMBINANT plasmid o the most common in the tube is bacteria without plasmid o middle common is the bacteria with plasmid without insert o least common is the bacteria with the plasmid with the insert plasmid containing the gene is RECOMBINANT plasmid this is what we want but we have bacteria of all three types mixed together, we need a way of sorting it out step 7 o use selective medium in a process called BLUE-WHITE SCREENING o the media you make has to contain agar, water, nutrients to support the life of the bacteria, in addition to
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3.27.07 - 3.27.2007 Needed some other molecule of DNA for...

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