lab 5

Question 1

what is the mini prep procedure?

Answer 1

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

Question 2

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

Answer 2

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

Question 3

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

Answer 3

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

Question 4

by what principle does gel filtration chromatography separate molecules?

Answer 4

size and shape

Question 5

by what process does a bacteria acquire a foreign plasmid?

Answer 5

transformation

Question 6

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

Answer 6

a vector. the vector in this case is the plasmid

Question 7

what are the 2 types of plasmid replication?

Answer 7

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

Question 8

why is inserting genes into cells a useful practice?

Answer 8

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

Question 9

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

Answer 9

because the gene code is univresal

Question 10

what are the components of the gel filtration chromatography experiment?

Answer 10

matrix, chromatography column and the elution buffer

Question 11

what part of the chromatography matrix actually performs the separation?

Answer 11

the matrix (the beads)

Question 12

what is the stationary phase of the chromatography?

Answer 12

the matrix (beads)

Question 13

what is the mobile phase of the chromatography?

Answer 13

elution buffer

Question 14

what does the column of the chromatography matrix consist of

Answer 14

consists of a tube with a frit and elution spout

Question 15

what’s the frit?

Answer 15

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

Question 16

what does the elution buffer do?

Answer 16

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.

Question 17

what is recombinant DNA?

Answer 17

two sources of DNA together

Question 17

what is bigger RNA or DNA?

Answer 17

DNA

Question 18

what does the matrix consist of?

Answer 18

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.

Question 19

how does gel filtration chromatography separate molecules?

Answer 19

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

Question 20

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

Answer 20

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

Question 21

how does agarose gel electrophoresis separate molecules?

Answer 21

on the basis of their size and shape

Question 22

what weighs more, plasmid DNA or RNA?

Answer 22

plasmid DNA

Question 23

how does the agarose gel separate molecules?

Answer 23

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

Question 24

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

Answer 24

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

Question 25

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

Answer 25

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

Question 26

true or false, supercoiled plasmids migrate faster than RNA?

Answer 26

false, RNA migrates faster

Question 27

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

Answer 27

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

Question 28

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

Answer 28

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

Question 29

what is the void volume?

Answer 29

volume of buffer between the beads

Question 30

what is the bed volume?

Answer 30

volume of beads + void volume

Question 31

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

Answer 31

-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

Question 32

Identify the nicked open/supercoiled DNA and the RNA

Answer 32

Question 33

What will this look like?

Answer 33