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What is 1 + 3?
pGlo Transformation Lab Test
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