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create multiple copies of the transformed bacteria

purpose of lesson 2

luria broth: growth
amp: shows that bacteria has GFP, which prevents ampacillin from killing the bacteria
arab: make everything glow

solutions used in lesson 2

concentrate the bacteria and release the contents of the bacteria including GFP and protein

two main purposes of lesson 3

to create a pellet or colony clump of bacteria

purpose of using a centrifuge in lesson 3

the density of the bacteria causes it to drop to the bottom of the column

what property of the solutions does a centrifuge use to cause separation?

pellet: multiple clumped bacteria colonies
supernatant: lb/amp/ara solution

two resulting portions after centrifuge in lesson 3


solution needed to continue with lesson 3

lysozyme: enzyme that breaks down the cell wall and allows contents to flow out

what is the enzyme used in this lesson? what does it do?

to burst the cell and then defrost it, which will pour bacteria's contents out

what was the purpose of freezing the contents of the tube at the end of this lesson?

1) separate bacterial debris from solution of proteins
2) prepare the column for binding
3) denature proteins using BB to make them stick to the column

three major objectives of lesson 4

to isolate the pellet

purpose of centrifuge in lesson 4

supernatant: has the proteins
pellet: not needed

two portions after centrifuge in lesson 4

BB: has high salt concentration and denature proteins and makes it hydrophobic and stick to the column

purpose of BB and its purpose

final separation of proteins

purpose of lesson 5

HIC: hydrophobic interaction chromatography

process used to separate proteins in lesson 5

plastic container, had microscopic hydrophobic beads

description of column

WB: renature bacterial proteins, now hydrophylic proteins fall off of the beads and into the test tube, GFP still sticks to the column

wash buffer's purpose in lesson 5

TE buffer: low salt concentration, goes into tube 1

TE buffer purpose in lesson 5

tube 1: TE
tube 2: andogenous regular bacteria
tube 3: GFP

contents in three tubes at the end

yes, it glowed

were you successful? how did you know?

separate any human protein made by bacteria for pharmaceutical use

real life application of this type of process

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