Mini Prep Flashcards
What is plasmid?
A double stranded, circular extra chromosomal DNA of bacterium
What is plasmid used for?
In recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA
What are the three steps of purifying plasmid DNA?
Growth of bacterial culture
Harvesting and lysis of bacteria
Purification of plasmid
How is alkaline lysis used?
Breaks the cells open to collect cell components
Outline the alkaline lysis method
Bacteria containing plasmid grown
Lyses with buffer containing SDS and NaOH
Through agitation, precipitation, centrifugation and the removal of supernatant, the plasmid is isolated
What does the SDS do in solution II?
Denatures cell proteins
Cleaves the phospholipid bilayer of the membrane
What does the NaOH do?
Disrupts hydrogen bonding between DNA bases which converts dsDNA to single stranded DNA
Also breaks down cell wall
Describe the re-suspension of bacterial pellet in alkaline lysis
Resuspend pellet in solution containing tris, EDTA, glucose and RNase A - normally called solution I
What are the roles of the components of solution I?
EDTA - chelates Mg+ and Ca2+ to stop DNAase damaged the plasmid. Also destabilises the cell wall
Tris HCl - buffer
Glucose - maintains osmotic pressure so cells don’t burst
RNAase A - degrade cellular RNA when cells are lysed
Explain the neutralisation with solution III in alkaline lysis
Addition of potassium acetate (solution III) to decrease the alkalinity of the mixture
plasmid DNA re-natures to dsDNA here
This dissolves in solution whilst everything else becomes a white precipitant
How can you separate precipitant from solution?
Centrifugation
Outline the cleaning and concentration in alkaline lysis
Clean out salts, EDTA, residual proteins, debris and RNase
Ethanol precipitation
What is LB broth?
Nutrient rich medium which is primarily used for growth of bacteria
Usually includes peptides; casein peptones; trace elements and minerals
How can bacteria be selectively grown?
Introduction of an antibiotic
In this instance it was kanamycin which was specific to the plasmid
What observation can be made with LB broth when bacteria successfully cultures?
Gets more cloudy as bacteria grows
Explain what the following component of the lysis buffer is for: tris hcl
Act as a buffer to maintain a stable pH
Explain what the following component of the lysis buffer is for: Sucrose
Create an oncotic pressure
Explain what the following component of the lysis buffer is for: EDTA, EGTA
Chelate divalent ions to prevent unwanted reaction
Explain what the following component of the lysis buffer is for: sodium fluoride and sodium pyrophosphatase
Protease inhibitors to prevent enzymatic breakdown of the protein
Explain what the following component of the lysis buffer is for: Triton X
Detergent
What are the components of the lysis buffer?
Tris HCl Sucrose EDTA EGTA Sodium fluoride Sodium pyrophosphatase Triton X
What are the advantages of using a lysis buffer?
Makes protein extraction simpler
Done without specialised equipment
Done at room temp on desk
What are the disadvantages of using a lysis buffer at room temperature?
Room temp may cause degradation of the protein
What does this method remove from the pellet and leave you with?
Removes protein and chromosomal DNA
then salts and small molecules at alcohol precipitation
Then leaves you with plasmid DNA
Explain the roles of the components of solution II?
NaOH - disrupts H bonding in the dsDNA bases to convert to ssDNA
SDS - Detergent. Denatures proteins in cells and solubilises the membrane
Explain the roles of the components of solution III
K acetate - decreases alkalinity to allow renature of DNA
Glacial - Ph balance
Water - dsDNA can dissolve in water but genomic DNA cant. Helps denatured proteins clump together.
What can be used instead of isopropanol to washed the DNA pellet?
Phenylchloroform
Why is isopropanol used > phenylchloroform?
Nasty burns
