Transformation Lab
When should you sterilize the metal inoculating loop?
Before you transfer isolated colonies to the +/- plasmid tubes and after you transfer them
How long should they be incubated at room temp and in an incubator?
24-36 hours at 37 degrees celcius in an incubator or 48-72 hours at room temo
What is then done next?
Add Luria broth (LB) to each tube and let rest in test tube rack for about 15 minutes
What do you add to the tubes after adding cells? How do you mix the DNA with the cell suspension?
Add the pgreen plasmid to +plasmid tube; micropipette or flick with finger
Be careful not to transfer any what from the plate along with the cell mass?
Agar
What is a positive control?
An experiment that shows that nothing has gone wrong in the procedure: a positive result is expected
What do you add to the tubes before you add bacterial colonies? Then what do you put the tubes on?
CaCl2; ice
What is the next thing to calculate? How do you figure that out?
Calculate the total volume of cell suspension prepared; look at how much CaCl2, DNA, and broth was added: 250, 10, and 250 = microliters of that all
What is the last step then?
Calculate transformation efficiency: colonies observed/mass of plasmid in cell suspension spread = 9/9.8x10^-3 = 918.4 colonies per microgram of plasmid DNA
Transformation is limited only to what cells? Because of this fact, does increasing the amt of plasmid used increase the probability that a cell will be transformed?
Cells that are competent; not necessarily
The object is to determine what?
Determine the mass of plasmid that was spread on the experimental plate and that was, thus, responsible for the transformants (number of colonies) observed
What is the first step in finding the transformation efficiency?
Finding the total mass in microgram of plasmid used
How many plates should we have and what should they be labeled?
Four 1) LB + plasmid 2) LB - plasmid 3) LB/amp - plasmid 4) LB/amp +plasmid
What should we do to the tubes while they are in the water bath? What do we do after we heat shock them?
Gently agitate them; return them to ice for one or more minutes
The plasmid contains what genes and marker?
It contains pGreen (GFP) gene and ampicillin resistance gene and a fluorescent marker
Why do we add CaCl2?
It helps the lipid bilayer of the cells to open up to the plasmid DNA but we still need to heat shock them and that's why we need to put them on ice right away after heat shock so the wholes don't stay open forever and all the inner contents of cell fall out
What does the LB/amp - plasmid plate stand for?
It is a negative control
What is a negative control?
It is an experiment where a negative result is expected to be yielded
Is the GFP gene normal or mutant?
It is mutant so that the bacteria will glow under UV light
What does the phenotype of the transformed colonies tell you?
It tells us that those bacteria have successfully been transformed with the plasmid because they grow under UV light indicating they received mutant form of GFP gene and grow on ampicillin so they got the ampicillin resistant gene
How was the transformation made possibble?
It was made possible by heat shocking the bacterial cells in order to make them competent to pick up the plasmid
What are the positive control plates in this experiment? What do these plates test for?
LB + plasmid and LB - plasmid; they test for the viability of the cells after they have gone through the transformation procedure
After adding plasmid what do we do?
Let the +/- plasmid tubes incubate on ice
What do you do when you add the cells to the plate? How should the glass beads not be moved?
Place 4-6 glass beads through the clam shell method and use a back and forth and up and down shaking motion; they shouldn't be moved swirling around and around
Then what is calculated next and how do you do that?
The total mass of plasmid in the cell suspension spread: total mass x fraction spread = 0.05 microgram x 0.196 = 9.8 x 10^-3 microgram
Describe the appearance of the LB+ plasmid plate?
Same as answer in last flash card
A sample of competent cells is usually _________ with what? Excess DNA may actually interfere with what?
Saturated with the addition of a small amt of plasmid; the transformation process
Moving these glass beads around like that helps with what?
Spreading the cell suspension all over the agar surface
In order to see if it is milky white, what should we compare this tube to?
The tube containing CaCl2 which will be a clear solution
What is added to the +plasmid tube? What does the -plasmid tube serve as?
The DNA plasmid; serves as the control
Then what is done?
The cells are spread on the deisgnated plates
Then what is calculated? What is the equation for that?
The fraction of the cell suspension that was spread on the plate: volume suspension spread/total volume of cell suspension = 100/510 = 0.196
How is transformation efficiency expressed?
The number of antibiotic-resistant colonies per microgram of plasmid DNA
Which plate is the experimental plate?
The plate with LB/amp +plasmid
What is the purpose of this experiment?
The purpose is to successfully transform the Escherichia coli bacteria when we expose it to extracellular plasmid DNA that contains the pGreen gene and the gene for ampicillin resistance.
The total area of the colonies picked should be equal in size to what?
The tip of a pencil eraser
Describe the appearance of the LB - plasmid plate? What does this control tell us?
There is going to be a lawn of bacteria seen on the plate; this tells us that the bacteria remained viable after undergoing the transformation procedure
Describe the appearnce of the LB/amp - plasmid plate? What does this control tell us?
There was no bacteria growth seen because the plate has ampicillin on it and since these bacteria were not transformed with the plasmid containing amp resistant gene they will not grow on the plate; this confirmed that ampicillin did not allow any growth of Escherichia Coli if it dd not contain the plasmid
Describe the appearance of the LB/amp + plasmid plate?
There were 9 colonies on the plate and they glow under UV light
Once this is done for both the +/- plasmid tubes, what is done with the plates?
They are placed upside down at room temp or in an incubator
What happens after we incubate these tubes on ice?
We heat-shock them in a 42 degree celcius water bath for 90 seconds or more
When finished spreading with glass beads, what do we do?
We let the plates rest so the cell suspension can sink into the agar
What were you selecting for in the experiment? (what allows you to identify which bacteria have taken up the plasmid?
We selected for a plasmid that contains a gene for ampicillin resistance so that after transformation the bacteria that took up the plasmid can be distinguished fro mthose that did not by plating the bacteria on a medium containing ampicillin
How do you remove the glass beads?
You hold it vertically over garbage and clam bottom portion of plate and tap on glass beads to come out
How do you mix the cells in the tube with the plasmid? What color should the suspension look like?
You micropippette the solution in and out several times; milky white
How do you find the total mass in microgram of plasmid used?
total mass = volume x concentration; we added 10 microliters of plasmid to the tube at a concentration of 0.005 microgram/microliter: (10 microliters)(0.005 microgram/microliters_) = 0.05 micrograms
