Practical 1 Flashcards
(34 cards)
What is the range of a p2 pipette?
0.2-2uL
what is the range of a P20 pipette?
2.0-20.0 uL
what is the range of a P200 pipette
20-200uL
what is the range of a p1000
100-1000uL
describe the steps for experiment 1
1) cut open circular DNA plasmid with restriction enzyme
2) ligate foreign DNA into plasmid vector
3) transform Ecoli host with recombinant plasmid
4) isolate transformed hosts on antibiotic plates
5) isolate recombinant plasmid from host bacteria
6) perform restriction enzyme digests on recombinant plasmid
7) Analyze DNA fragments by agarose gel electrophoresis
why do we need to open the circular plasmid DNA with a restriction enzyme
we need to insert the foreign PCR DNA product to make recombinant DNA
how does ligase work in the formation of a recombinant DNA
ligase forms phosphodiester bonds between the phosphate sugar backbones of the DNA insert and plasmid
what is transformation
transformation is a process where bacteria spontaneously “take up” plasmid DNA from surrounding environment
what is a plasmid? where are they found
a plasmid is a small double stranded circle of DNA that is found in some bacteria in addition to their larger circular genome
they carry extra information not required for the survival of a cell
what is the purpose of the origin of replication in a plasmid? What would happen without it?
it allows it to be replicated alongside the host DNA, without it, the plasmid would be lost
what is the purpose of the cloning site, what would happen without it
the cloning site are recognition sequences for the restriction enzymes to cut at
without it, we would not be able to open the plasmid and insert the DNA
what is the purpose of a selectable marker? what would happen without it
a selectable marker allows us to select only transformed cells
without it, we would not be able to differentiate which cells were transformed and which were not
what is molecular cloning
it is the process of inserting a piece of foreign DNA into a plasmid vector and recovering it in colonies of bacteria that are identical in their DNA as they came from the same ancestral cell
how does ligase work? what type of bond does it form
ligase creates a phosphodiester bond between the 3’ OH end of a sugar and a 5’ phosphate group in order to seal the phosphate sugar backbone of DNA
how/why does incubation with calcium chloride increase the competency of E coli cells
the calcium ions act to neutralize the negatively charged cell membrane and allow the negatively charged DNA to bind to the surface of the bacterial cell surface and then can enter during a heat shock
what does it mean to increase a cell’s competency?
it means to make a cell membrane more permeable and foreign DNA can enter the membrane more easily
why did we need to add fresh media after transformation and incubate the Ecoli for 40 minutes at 37C
The bacteria need to grow and repair themselves after the transformation. They also need time to express their antibiotic-resistant gene. The temperature and nutrient rich broth aid in this recovery process
What are the steps in the plasmid miniprep protocol?
1) resuspend cells
- allows even lysis buffer
2) add lysis buffer
- has chaotropic reagents
3) add neutralization and form precipitates
4) spin down debris and collect clear supernatant
5) apply to column
6) wash 2x with ethanol solutions to remove excess debris
7) dry column
- removes excess ethanol and prevents dilution of elution buffer
8) elute DNA off column
what is a chaotropic reagent? what would happen if we didn’t include them
the chaotropic reagents allow us to bind the DNA to the silica membrane by interrupting the hydrogen bonding between DNA and water. Without it, the DNA would not stick to the membrane and could not be purified
what would be the problem if we eluted our plasmid DNA immediately after applying the cleared lysate to the column and allowing it to bind
if we elute the plasmid without washing it, it is likely that the eluted DNA will not be pure and may have excess buffer remnants or cell components mixed in it
what is the “big picture” of digesting plasmid preps with EcoRI and NsiI
the plasmids have been engineered to have two cut sites for these enzymes that surround the insert itself. By using these enzymes we can remove the insert and see if there are any other cut sites within the insert itself.
By comparing cut site differences among the different PCR inserts, we may be able to conclude that these PCR inserts came from different species of bacteria
why do smaller fragments migrate though the gel more quickly
the concentration of the gel causes the pores to be larger or smaller, for higher concentrations, the pores are smaller and can slow down larger fragments of DNA
why do tight conformations of DNA migrate through the gel slower?
they are more compact and experience less friction as they travel through the pores of the gel
what is the purpose of including a “ladder” on gels
it allows us to determine the size of linear DNA fragments by comparing their migration patterns to the patterns of DNA fragments of known sizes
