lab 5 Flashcards

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1
Q

what is the mini prep procedure?

A

The miniprep procedure separates plasmid DNA and some bacterial RNA from the bacterial chromosomal DNA, bacterial proteins and membranes.

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2
Q

what do we do after the mini prep procedure to further purify plasmid DNA?

A

Because the miniprep still contains bacterial RNA, it can be further treated with RNAse which degrades the remaining RNA without affecting the plasmid DNA. However, when large amounts of purified plasmid DNA are required, it can be separated from bacterial RNA by GEL FILTRATION CHROMATOGRAPHY

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3
Q

what are the 2 techniques used in this lab so separate plasmid DNA and bacteria RNA

A

In this lab you will be given a mixture of plasmid DNA and RNA which will first be separated into fractions using GEL FILTRATION CHROMATOGRAPHY. The fractions you collect from this procedure will then be further separated using AGAROSE GEL ELECTROPHORESIS. The combined procedures will result in a clearer separation of the nucleic acids

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4
Q

by what principle does gel filtration chromatography separate molecules?

A

size and shape

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5
Q

by what process does a bacteria acquire a foreign plasmid?

A

transformation

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6
Q

what do we need in order to introduce a foreign gene into a cell? what is it in this case?

A

a vector. the vector in this case is the plasmid

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7
Q

what are the 2 types of plasmid replication?

A

-stringent: occurs with the bacterial chromosome (one copy per cell)
-relaxed: occurs autonomosly (multiple copies per cell (10-500))

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8
Q

why is inserting genes into cells a useful practice?

A

-clone a gene to use in gene therapy
-encourage the cell to translate it into the gene product

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9
Q

how does the bacteria replicate the human gene once its in the bacterial cell?

A

because the gene code is univresal

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10
Q

what are the components of the gel filtration chromatography experiment?

A

matrix, chromatography column and the elution buffer

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11
Q

what part of the chromatography matrix actually performs the separation?

A

the matrix (the beads)

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12
Q

what is the stationary phase of the chromatography?

A

the matrix (beads)

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13
Q

what is the mobile phase of the chromatography?

A

elution buffer

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14
Q

what does the column of the chromatography matrix consist of

A

consists of a tube with a frit and elution spout

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15
Q

what’s the frit?

A

a membrane or porous disk that supports and retains the matrix in the column but allows water and dissolved solutes to pass

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16
Q

what does the elution buffer do?

A

The column, with the matrix and applied sample, is “developed” by the elution buffer. This means that the molecules in the sample are carried by the flow of buffer into the matrix where they are gradually separated.

17
Q

what is recombinant DNA?

A

two sources of DNA together

17
Q

what is bigger RNA or DNA?

A

DNA

18
Q

what does the matrix consist of?

A

consists of microscopic beads that contain pores and internal channels. The larger the molecule, the more difficult it is for it to pass through the pores and penetrate the beads.

19
Q

how does gel filtration chromatography separate molecules?

A

the larger, higher molecular weight molecules are eluted from the column before the smaller ones. Larger molecules take the faster, more direct path that involves less time in the beads

20
Q

when do we conduct agarose gel electrophoresis? what is the point of it?

A

after gel filtration chromatography. will be used to analyze and further separate the RNA and DNA after chromatography

21
Q

how does agarose gel electrophoresis separate molecules?

A

on the basis of their size and shape

22
Q

what weighs more, plasmid DNA or RNA?

A

plasmid DNA

23
Q

how does the agarose gel separate molecules?

A

The agarose gel has microscopic pores that act as a molecular sieve.

24
Q

what are the different forms of plasmid DNA? which travels faster?

A

Supercoiled plasmid DNA, which is coiled on itself, is the most compact form and travels farther than nicked open, circular plasmid DNA.

25
Q

why do fragments migrate in the agarose gel sample? where do they migrate to?

A

Since nucleic acids have a strong negative charge at neutral pH (due to the phosphate groups), they will migrate through the gel towards the positive electrode in the presence of an electric field

26
Q

true or false, supercoiled plasmids migrate faster than RNA?

A

false, RNA migrates faster

27
Q

what is the difference between gel filtration chromatography and agarose gel electrophoresis?

A

agarose gel electrophoresis: smaller molecules move faster
gel filtration chromatography: smaller molecules move slower

28
Q

which one is better, agarose gel electrophoresis or gel filtration chromatography?

A

gel electrophoresis possesses greater separation power and is well suited for the analysis of very small quantities of nucleic acids.

29
Q

what is the void volume?

A

volume of buffer between the beads

30
Q

what is the bed volume?

A

volume of beads + void volume

31
Q

what is the link between void volume and the elution of large molecules?

A

-Large molecules that do not enter beads will elute at the void volume

-Ex: if void volume is 20 drops, largest molecules will begin exiting the column starting from drop 21

32
Q

Identify the nicked open/supercoiled DNA and the RNA

A
33
Q

What will this look like?

A
34
Q
A