Theory Exam 2 Flashcards
You have a tube that contains a bacterial culture that has 10,000 CFU/mL.
If you did not perform any dilutions, how many colonies should grow from:
* 1 mL
* 10 μl
1 mL: 10,000 colonies
10 μl: 100 colonies
You have a tube that contains a bacterial culture that has 10,000 CFU/mL.
You do a 1 in 100 dilution. What is the concentration of the bacterial culture in the dilution blank in CFU/mL
100 CFU/mL
You have a tube that contains a bacterial culture that has 10,000 CFU/mL.
You do a 1 in 100 dilution. How many colonies will grow if you plate 1 mL?
100
You have a tube that contains a bacterial culture that has 10,000 CFU/mL.
You do a 1 in 100 dilution. How many colonies will grow if you plate 200 μl?
20
A student prepares a 10–6 dilution of a broth culture and plates 0.1 ml in a spread plate and 0.2 ml in a pour plate. After 48 hours of incubation, the student counted 42 colonies on the spread plate and 124 colonies in the pour plate. The student thinks there might be something wrong with their assay. Given that the same
dilution blank was used for both plates, the student expected to see approximately 84 colonies
in the pour plate since twice the volume was plated relative to the spread plate. Alternatively,
the student might have expected 62 colonies on the spread plate since half the volume was
plated relative to the pour plate.
Suggest reasons why the numbers don’t seem to match up
- Some bacteria may have been killed by a hot spreader.
- Some bacteria may have been killed by ethanol if the spreader was not flamed.
- Some bacteria were still stuck to the spreader and not transferred to the plate.
- Pipetting errors.
What is the concentration
of the original bacterial sample?
Plate A: 4209 | 3986
Plate B: 296 | 280
Plate C: 31 | 32
Plate D: 26 | 29
A student has a culture with a density of 7.0 x 109 cells/ml and wants to do a pour plate assay.
She wants to have 75 colonies on the plate after plating 0.5 ml.
Sketch out a dilution scheme
that the student could use to achieve this goal.
While working in a lab, your mentor asks you to isolate some plasmid. The first thing you do is set up an overnight culture in L-broth. How do you proceed to purify the plasmid?
- Centrifuge cells, pour off L-broth, keep the cell pellet
- Add resuspension buffer to resuspend cells
- Add NaOH and SDS to lyse the cells
- Potassium acetate and ethanol solution to neutralize the mix
- Centrifuge the pellet cell debris and chromosomal DNA
- Put solution through the column; plasmid binds to resin
- Use the column wash solution
- Add Tris-EDTA solution to elute the plasmid
What is the critical step that allows you to separate plasmid DNA from chromosomal DNA? Why is this step effective?
- Addition of potassium acetate (KOAc) to neutralize the NaOH.
- Chromosomal DNA, due to its large size, cannot re-anneal and becomes trapped in the K/SDS
complex and can be centrifuged out. - The plasmid DNA is smaller and easily renatures upon addition of KOAc.
- The renatured plasmid remains soluble in the solution and can be found in the supernatant.
A student isolated a plasmid from E. coli and performed a restriction enzyme digest using three different restriction enzymes (as single digests). After the digest, the student used gel
electrophoresis to separate the DNA fragments. She used a linear DNA ladder so that she could
compare the sizes of the DNA fragments.
The DNA ladder is in Lane 5 and the uncut plasmid is Lane 1. Lanes 2 – 4 represent one of the bands from Lane 1.
- What are the three forms that a plasmid that exist in once it has been isolated from the bacterial cells and indicate which of Lanes 2 – 4 represents that form of the plasmid.
- Given no other info, and using no math, how big is the plasmid?
- Where on the gel (relative to the wells) would the positive and negative electrodes be? Put a
“+” and “–“ sign in the open circles by the gel drawing. Why are they placed in this orientation?
- Lane 2 - open circle
- Lane 3 - linear
- Lane 4 - supercoiled
- Approx. 6.56 kb
- should be on the bottom
- should be on the top
- DNA is negatively charged and will move to +ve electrode
