Lecture 2 - Lecture 2 Slide 5 Cell components can be...

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Lecture 2 Slide 5 Cell components can be separated by charge, size or affinity. Slides 6/7 Centrifuge If you break open a whole bunch of cells and you centrifuge them, you can separate out the organelles and you can separate molecules from each other. If you centrifuge cells in low speed, only big things settle to the bottom and if you take what is left (supernatant) and you spin it at a very high speed, smaller things settle to the bottom and if you keep doing this until what spins down is very large molecule. This is one of the first techniques used to separate out all the structures from cells. The problem is that there are lots of things inside of cell. Slide 8 Column Chromatography You can then separate molecules from each other by chromatography. There are essentially three different kinds of chromatography. You can separate things by affinity, size or charge. Slide 9 Example of separating things by affinity You have molecule X and you want to know all the molecules inside the cell that are associated with it. Now they have to associate in a very strong way, usually by more than van der waals forces. So what you can do is attach a tag ( GST ) to it and then we have a chromatography column and we fill it up with a material and we take molecule X and put it in the column and it sticks to the inside and now we break up cells and pour all the contents through the column. Everything that sticks to molecule X can be eluded later and how we find out 3 different guys associate with molecule X. This is a simple-minded way of separating molecules of the cell by affinity. Slides 10/11 You can also separate all the molecules (by size) by electrophoresis. Slide 12/13 There are many ways of inserting DNA into cells. Here, you wrap DNA around a vesicle and add it to cells and you rig the vesicle in a way where it recognizes the cell and merges with it and dumps the DNA on the inside. Another method is to take the DNA molecule and attach it to gold particles and then you just blast the gold particles at the cells (used to do it with guns, now uses high pressure). Some of the DNA blasted into the cell will get expressed. Another technique is to make cell membrane transiently permeable. You open up the membrane and allow DNA to get inside and the membrane close up again and the DNA hopefully will get expressed. Slides 14-16
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Neat thing about plasmid is that by using restriction enzyme, you can insert another gene to it, put it back together again and stick it inside another cell and it will be expressed. Restriction enzyme restricts the ability of viruses to proliferate inside the cells. Once you inserted the DNA inside a cell, you can grow the cell like crazy and then of course you really copied DNA with the gene that you inserted. Slide 18
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Lecture 2 - Lecture 2 Slide 5 Cell components can be...

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