Yeast lab Flashcards
what was the purpose of part 1 of the yeast experiment
isolation of yeast genomic DNA, restriction enzyme digest, and dephosphorylation of plasmid DNA
how was DNA isolated?
we extracted DNA from a yeast culture, and then used kits (with all the necessary buffers and columns) to isolate the DNA
what kit did we use to isolate DNA?
GeneJet Genomic DNA Purification kit
after lysing the cells, what did we treat them with?
RNase A
what does RNase A do?
removes the RNA
once the RNA was removed from our samples, what did we do?
used the purification column with its silica membrane
what is the purpose of the silica membrane in the column?
the DNA binds to the membrane
why do we wash the column with buffers?
to remove impurities
why don’t the buffers remove the DNA as well?
DNA is bound to the silica membrane
what is used to remove DNA from the column?
elution buffer
what plasmid was used as our cloning vector?
pUC19
what important things does pUC19 contain?
AmpR, ORI, and MCS
what is AmpR?
ampicillin resistance
what is ORI
origin of replication. Allows for replication in a bacterial host
what is MCS
a multiple cloning site that contains various unique restriction sites
how was pUC19 digested?
using the restriction enzyme EcoRI
what sequence does EcoRI recognize?
5’-GAATTC-3’
what does EcoRI do at the 5’-GAATTC-3’ sequence?
it makes a staggered cut between the G and the A to create complementary sticky ends
what is the final step of part 1?
dephosphorylation of the digested plasmid
how do we dephosphorylate the digested plasmid?
by using alkaline phosphatase
why do we dephosphorylate the digested plasmid?
to prevent recircularization of during the ligation reaction
what is the purpose of part 2 of the yeast experiment?
restriction enzyme digest of genomic DNA, ligation, preparation of competent E coli cells, transformation and plating
what does EcoRI do?
digests the isolated yeast genome into smaller restriction fragments
how do we digest the isolated yeast DNA into smaller restriction fragments?
by using EcoRI, the restriction enzyme
why do we digest the isolated yeast DNA into smaller restriction fragments?
it creates compatible sticky ends with the dephos./EcoRI-digested pUC19 from last week
how do we make a recombinant vector?
take the dephos./EcoRI-digested pUC19 with the EcoRI-digested yeast DNA in the presence of ligase
why is ligase present when creating a recombinant vector
DNA ligase will form the phosphodiester linkages at the junctions of the hybridized molecules
in this lab, which type of ligase did we use and why?
T4 ligase, because it’s the most active at room temperature
define transformation
the genetic alteration of a cell resulting in the uptake, integration, and expression of exogenous material from a cell’s environment
define competent cells
cells that are capable of being transformed
what are cells that are capable of being transformed called?
competent cells
what did we use to cause the bacterial cells to become competent?
T-solution
how does heat shocking aid in transformation?
it is used to assist in the uptake of exogenous material across the membrane. Fast moving molecules outside the cell will move into the colder regions inside cells
what strain of E coli cells were used?
JM101
how will we know if transformation has been successful?
any cell transformed with pUC19 plasmid will now be resistant to ampicillin, and will be white on a plate due to lacZ interuption
why will a transformed cell be white on a plate?
the lacZ gene of the MCS will be interrupted. LacZ produces beta-galactoside, which turns blue in the presence of X-gal
what were the transformed bacteria plated onto?
LB/Amp/X-Gal/IPTG
what does Amp on the plate do?
selects for the cells that were transformed with pUC19
what does X-Gal on the plate do?
it’s an artificial substrate of B-galactosidase, and will be hydrolyzed to produce a blue precipitate if the LacZ enzyme is present
what does IPTG on the plate do?
it’s a synthetic analog of lactose that inactivates the lac repressor and induces synthesis of B-galactosidase
what was the purpose of part 3 of the yeast experiment?
screening for E coli containing recombinant DNA
what is the formula for transformation efficiency?
(# colonies/ng of DNA plated) x 1000ng/ug
after isolating two white colonies from the plate, we added them into test tubes containing ___
LB/Amp
what is the purpose of part 4 of the yeast experiment?
isolation of plasmid DNA using the alkaline lysis method
what is the purpose of alkaline lysis?
it makes possible the isolation of individual plasmid clones from large numbers of individual bacterial colonies
what does alkaline lysis do?
it uses a high pH (12.1-12.5) to denature large DNA sequences. This allows the selective precipitation of bacterial genomic DNA leaving the smaller plasmid DNA in the solution
T or F: other components of alkaline lysis remove additional contaminating macromolecules, resulting in a relatively clean plasmid DNA sample
true
in the alkaline lysis method, how do we ensure we end up with a relatively clean plasmid DNA sample?
other components of the method remove contaminating macromolecules
T or F: as bacteria grow, they release cell wall components that may contaminate the final DNA preparation and inhibit enzyme action
true
because bacteria release contaminating cell wall components, why is it important we remove them
because they contaminate the final DNA preparation and inhibit enzyme action
do we remove the cell wall components of the bacteria before or after alkaline lysis?
before
describe the “resuspension of cells” component of alkaline lysis
cells are suspended in alkaline lysis solution 1, and each component of it has a specific function
what are the components of alkaline lysis solution 1?
Tris, EDTA, and RNase
what is the function of Tris in the alkaline lysis solution 1?
it buffers the solution, allowing cells to be resuspended in an isotonic solution
what is the function of EDTA in the alkaline lysis solution 1?
it’s a chelating agent that forms strong complexes with divalent metal cations. This inactivates DNases present in the cells and destabilizes the cell membrane
what is the function of RNase in the alkaline lysis solution 1?
it removes RNA molecules
T or F: before moving onto the “cell lysis” step of alkaline lysis, we want our solution to be clumpy
false! it is very important that the cells are completely resuspended and that there are no clumps left to ensure complete lysis in the next step
describe the “cell lysis” step of alkaline lysis
lysis is achieved by the addition of solution 2
what are the components of alkaline lysis solution 2?
sodium dodecyl sulfate, NaOH
what is the function of SDS in the alkaline lysis solution 2?
it’s a detergent that dissolves the components of cell membranes and denatures cytoplasmic proteins
what is the function of NaOH in the alkaline lysis solution 2?
it helps break up the cell wall, thereby freeing cellular contents into the solution. It also denatures DNA
what happens to DNA treated with alkaline lysis solution 2?
it becomes linearized and denatured whereas plasmid DNA remains circular
why does plasmid DNA remain circular after treatment with solution 2?
because it’s much smaller and supercoiled
describe the “neutralization of the reaction and clearing of the lysate” step of alkaline lysis
this step is accomplished with solution 3
what are the components of alkaline lysis solution 3?
KOAc (potassium acetate)
what is the function of KOAc in the alkaline lysis solution 3?
drops the pH to make it neutral, which causes the genomic DNA and proteins to aggregate and form an insoluble complex that precipitates due to the high concentration of salt
KOAc interacts with SDS, which precipitates out
the neutral pH allows the plasmid DNA to renature properly while genomic DNA is being precipitated out
describe the “precipitation and de-salting” step of alkaline lysis
a specific concentration of alcohol (ethanol) along with a salt that masks the changes on the DNA will precipitate DNA. In this case, ethanol and KOAc are used to precipitate the plasmid DNA
a further wash with 70% ethanol is performed to remove residual salt from the already precipitated plasmid DNA
what is the purpose of part 5 of the yeast experiment?
restriction enzyme digest of isolated plasmid DNA, and agarose gel electrophoresis
what is the purpose of a DNA size marker?
allows you to determine the sizes of the DNA fragments in your sample
what is a DNA size marker
a large piece of DNA that’s been digested with one or more restriction enzymes to produce a known array of DNA fragments that range in size
what was the purpose of part 6 of the yeast experiment?
isolation of sequencing grade plasmid DNA, and analysis of sequencing results
how do we purify our DNA
we use a kit that comes with premade buffers and a silica spin column. This enables the isolation of high quality plasmid DNA from E coli in a very short time period
T or F: the procedure first subjects cells to SDS/alkaline lysis
true
what happens to the resulting lysate?
it is neutralized and then centrifuged to pellet out cell debris and the SDS precipitate
why is the silica membrane washed
to remove any contaminants
what is the last step before sequencing?
the plasmid DNA is eluted in a small volume of buffer, and then sent in for sequencing
