Lab 6

Question 1

Agarose

Answer 1

a highly purified polysaccharide, used for preparing gels that separate DNA
or RNA molecules by size

Question 2

UV transilluminator

Answer 2

a highly-intensity ultraviolet light source that is used for visualizing
DNA stained with fluorescent dyes including ethidium bromide.

Question 3

Ethidium bromide

Answer 3

a commonly used DNA stain. Ethidium bromide binds to DNA. It
absorbs ultraviolet light, and gives off orange visible light that can be easily detected.

Question 4

Tris

Answer 4

a commonly used buffer in molecular biology. Tris has a pKa of 8.1 and a useful
buffering range between pH ~7-9

Question 5

EDTA

Answer 5

a metal chelator that is frequently added to solution containing DNA. EDTA
tightly binds metal ions like Mg2+ that are required cofactor for the activity of DNase
(enzymes that degrade DNA)

Question 6

TBE (Tris-borate) buffer

Answer 6

An electrophoresis buffer containing Tris, boric acid, and
EDTA

Question 7

TAE (tris-acetate) buffer

Answer 7

An electrophoresis buffer containing Tris, glacial
acetic acid, and EDTA. When recovering DNA from agarose gels, 1X TAE buffer is
recommended for electrophoresis

Question 8

DNA Loading buffer

Answer 8

A solution added to DNA before it is loaded into an agarose gel.
This buffer facilitates loading DNA by making the DNA solution much denser than the
electrophoresis buffer. The loading buffer also contains one or more dyes that permit
one to gauge the progress of Electrophoresis

Question 9

DNA ladder

Answer 9

A DNA ladder is a solution of DNA molecules of different lengths used in
agarose gel electrophoresis. It is applied to an agarose gel as a reference to estimate
the size of unknown DNA molecules. In addition it can be used to approximate the mass
of a band by comparison to a special mass ladder

Question 10

When DNAs are driven through the gel by an
electrical current…

Answer 10

small DNA fragments migrate more quickly through the pores
compared to large DNA fragments

Question 11

Electroendosmosis

Answer 11

EEO is a measure
of the number of fixed negative
charges covalently bound to
the agarose sugars

Question 12

DNA
is induced to migrate through
an agarose gel by the
application of an electrical
field. Since each….

Answer 12

phosphate group in the DNA backbone has a net negative charge of
about -2 at neutral pH, DNA will migrate toward to the red, positive pole (anode) in the
electrophoresis chamber

Question 13

Electrophoresis

Answer 13

refers to the movement of
charged particles across an electrical field

Question 14

The gel is run at…

Answer 14

constant voltage

Question 15

The rate at which the fragments
electrophorese through a porous matrix like agarose is…

Answer 15

proportional to their mass, and
inversely related to the log of the length of the fragment

Question 16

Make 1% agarose gel

Answer 16

0.7g agarose into a flask
Add 70 mL 1x TAE buffer
Microwave 1:15 - 1:30 minutes to make solution clean
Put hot agarose liquid into gel box and wait for it to become solid (15-20 minutes)
Carefully remove the comb

Question 17

Running a DNA gel

Answer 17

Add DNA loading buffer into digested reactions

Load the gel (put box in chamber, fill with TAE, etc.)

Add 15 uL DNA ladder into the first well

Load DNA samples about 30 uL per well

Run gel 140 V for 30 minutes

After running gels, keep them in Ethidium bromide solution for 15-20 minutes

Take a picture under UV transilluminator

Question 18

Cutting DNA fragments from agarose gel

Answer 18

Lay gel on plastic membrane
Cut out the bands
Add 1mL QG buffer into each tube containing the gels