General Knowledge
Name That Buffer
Are you an enzyme, because I like the way you break it down!
Miscellaneous
100
True/ False: Qiagen genomic columns are used for DNA purification.
True!
100
In what order do you use the following buffers: (a.) High-salt buffer (b.) Low-salt buffer (c.) Medium-salt buffer
(b.) Low-salt buffer (c.) Medium-salt buffer (a.) High-salt buffer
100
What does RNase do in the reaction?
RNase degrades RNA!
100
Why is it bad to expose your sample to DNase during the purification process?
Because DNase will degrade DNA.
200
Why do you transfer the column into a new collection tube at one point in the procedure?
So that the eluting genomic DNA isn't contaminated.
200
Which buffer do you use to elute the DNA from the column?
The high-salt buffer (buffer QF)
200
Name two enzymes that degrade Staphylococcus cell walls.
Lysozyme and lysostaphin
200
What two things are done to avoid DNase contamination?
Adding ETDA (in lysis buffer) and wearing gloves!
300
Why can't you apply a cloudy solution to the Qiagen column? Why is the solution cloudy?
A cloudy solution will clog the Qiagen column. It is cloudy because the cells are not lysed.
300
What is the low-salt buffer used for?
To bind DNA to the resin in the column.
300
What is the purpose of adding protease to the DNA mixture?
Protease degrades enzymes.
300
What is the purpose of adding Isopropanol precipitate?
Isopropanol precipitate concentrates the DNA and removes salts from the solution.
400
What's the benefit of using Qiagen columns?
We don't have to use hazardous solvents. It's efficient for cell lysis (i.e. S. aureus, bacteria with thick cell wall).
400
What does the medium-salt buffer remove?
It removes RNA, proteins, and carbohydrates.
400
How does lysostaphin break down cell wall components?
Lysostaphin cleaves the pentaglycine bridge of peptidoglycan.
400
Name several methods of lysing cells.
Adding detergents, freeze-thaw, physical shearing, specific solvents, etc.
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