11. What things do we always do to prevent contamination of our cultures and reagents?
Use good aseptic technique: spray bench thoroughly with disinfectant before and after lab, wash your hands at the beginning of lab before handling anything
22. Why do we use E. coli for producing large amounts of plasmid DNA?
It has about a 20 minute generation replication time
33. Why do we always want to start an experiment with an isolated colony of E. coli?
It ensures a pure culture of genetically identical bacteria
44. What is transformation, and what is the major barrier to overcome in achieving it?
Transformation is the uptake of exogenous DNA by cells/ the major barrier is the cell membrane (due to the charges)
55. What are divalent cations, and why do we need them for our heat shock transformation?
Divalent cations are 2+ charged ions and they neutralize the negative charges of DNA and the phospholipid bilayer
66. You used plasmid DNA for your transformation. How is plasmid DNA different from other DNA?
It is a small and circular piece of extrachromosomal and replicate independently
77. How does heat shock facilitate the uptake of DNA?
Going from cold to hot to cold to hot destabilizes the membrane so the plasmid can get across
88. Why do we let cells incubate in media with no antibiotics for an hour before plating on media containing antibiotics overnight?
The cells need time to absorb, replicate, and express antibiotic resistance before coming into contact with the antibiotics
99. What are the major differences in the antibiotics: ampicillin and kanamycin?
Ampicillin inhibits bacterial cell wall synthesis and kanamycin disrupts protein synthesis
1010. What is the mechanism of action of ampicillin in preventing bacterial growth?
Inhibits enzymes responsible for cell wall synthesis
1111. What is the mechanism of action of kanamycin in preventing bacterial growth?
Binding to the 30s ribosomal unit causing a misreading of tRNA
1212. What is the name of the gene that confers, and the mechanism of resistance to ampicillin?
Betalactimase inhibts, ampicillin has a betalactamerin that cuts the enzyme so inhibition is inhibited
1313. How do you know if cells are resistant to ampicillin or kanamycin?
Whether or not it grows in the presence the antibiotic
1414. Why do colonies on ampicillin plates have smaller satellite colonies, but colonies on kanamycin plates do not?
Betalactimase is secreted so it diffuses away from the colony and begins ampicillin so non resistant colonies can grow
1515. Why do you put antibiotic in your overnight culture tube for plasmid purification the you have already selected for cells carrying the plasmid on the plate containing media with antibiotic?
You need to ensure continued selection of only the cells carrying the antibiotic resistance
1616. What are signs that a microcentrifuge is not balanced? Be able to indicate a balanced configuration of tubes on a drawing of microcentrifuge rotor.
The centrifuge will start rocking on the table/ tubes need to be evenly spaced
1717. After cells are collected by centrifugation and supernatant media is discarded, the cell pellet is resuspended in buffer P1 (aka resuspension buffer) which has two primary functions. What are those two functions of buffer P1?
1) Resuspends pellet so they can be lysed by SDS, 2) contains RNase
1818. Buffer P2 (aka lysis buffer) contains what substance to lyse the E. coli cells?
Sodium dodecyl sulfate (SDS)
1919. What is the best description of the type of reagent that substance in Buffer P2 is?
Strong, ionic detergent
2020. What does that substance in Buffer P2 do to proteins?
Denatures the proteins
2121. How do you know that the cells have been lysed?
The solution will be noticeably clearer once the cells are broken up
2222. Why do you only rock the tubes gently to mix from this point forward?
You don’t want to break up the gDNA so that it would not be removed from solution after addition of neutralization buffer
2323. In terms of pH, how would you describe the conditions of the contents of the tube through the addition of lysis buffer?
Alkaline with a pH greater than 7
2424. What is the function of Buffer N3 which is added next?
Neutralize solution back to physiological pH
2525. The addition of Buffer N3 results in the immediate formation of a precipitate made mostly of what?
Denatured proteins
2626. What other molecules should be removed from the solution by formation of this precipitate?
Large genomic DNA
2727. The precipitate and supernatant are separated by centrifugation. What biological molecules are still dissolved in the supernatant?
Plasmid DNA, lipids, amino acids, carbohydrates, nucleotides, major biomolecule monomers, but no RNA
2828. How is the plasmid DNA separated from the other biomolecules?
Centrifugation at maximum speed for 10 minutes
2929. When the column is washed with buffer PE, what prevents the plasmid DNA from being washed away with the residual unwanted molecules?
PE is mostly ethanol and DNA doesn’t like to be in ethanol, if you want DNA to precipitate you would put it in a high concentration of ethanol
3030. After spinning the columns with buffer PE and discarding the flowthrough, why are the empty columns centrifuged again before eluting?
To get all the ethanol off the pad
3131. Be comfortable appropriately using the following terms: lysate, supernatant, flow-through, eluate/elution.
Lysate: a fluid that contains the contents of a cell that has been broken down or lysed
Supernatant: the liquid portion of a sample that remains after centrifugation
Flow-through: the liquid that passes through a designated area without being retained or absorbed
Eluate/elution: the solution that contains the extracted molecule after the elution process is complete/ the process of extracting a specific molecule from a solid substance by washing it with a solvent
3232. Purified plasmid samples were analyzed on the Nanodrop instrument. What was determined by this analysis and how was it determined?
The nanograms per microliter of DNA (concentration)/ determined by UV absorption
3333. Given a known concentration of a substance dissolved in aqueous solution, you should be able to determine the volume of that solution needed to achieve a specific amount of the substance dissolved
C1V1 = C2V2
3434. Given a known concentration of a substance dissolved in aqueous stock solution, you should be able to describe how to dilute that stock solution to the concentration needed for a procedure.
Dilute with DEPC H2O
3535. Your purified plasmid was incubated with restriction enzymes.
a. What do these enzymes do?
b. Which restriction enzymes did you use?
c. What is a general characteristic of the DNA sequences relevant to the function of these enzymes?
d. How many times do these sequences occur in pAMP?
e. How many times do these sequences occur in pKAN?
Cut DNA
EcoR 1, BAMH1, HindIII
Sequences are palindromic
1
1
3636. After treatment with restriction enzymes, the plasmid DNA was analyzed by gel electrophoresis.
a. By what characteristic does gel electrophoresis typically separate molecules?
b. You ran a sample of untreated plasmid DNA on the gel. What three forms of plasmid DNA may be observed on the gel and what do the relative amounts of these forms tell you?
By size
Supercoiled, linear, relaxed circle
3737. If given a plasmid map, you should be able to tell how many bands you expect to observe when…
a. the plasmid is incubated with as specific restriction enzyme
b. the plasmid is incubated with any combination of specific restriction enzymes
yes, yes
3838. You also ran a sample of DNA treated with EcoRI and HindIII.
a. What is lamda?
b. What will this sample allow you to do with the bands in other lanes of your gel?
A bacteriophage, we know the sequence of the lamda genome, we know if we cut with certain enzymes how long it will be
can use this standard to estimate size of bands
3939. In considering a restriction digested plasmid of known total size run on a gel with a standard, how should the lengths of the fragments relate to the length of the undigested plasmid?
All fragments lengths should add up to the total undigested plasmid.