Lab Chapter 4: Part Two

Question 1

Describe the PPE required for this week’s lab

Answer 1

Nitrile gloves

Question 2

What does the 3D structure of restriction enzymes allow them to do?

Answer 2

attach to a double stranded DNA molecule and slide along until they recognize a sequence of base pairs.

Question 3

What will a restriction enzyme do if there is multiple restriction sites in a DNA molecule?

Answer 3

It will cut the DNA molecule into multiple fragments

Question 4

What determines the length of each fragment?

Answer 4

depends on the location of restriction sites in DNA molecule

Question 5

What does agarose gel elctrophoresis do?

Answer 5

When restriction enzymes cut multiple fragments, they can be separated and visualized with this process.

Question 6

What does the term electrophoresis mean?

Answer 6

to carry with electricity

Question 7

How does electrophoresis separate DNA fragments?

Answer 7

According to their relative size

Question 8

Describe general process of electrophoresis

Answer 8

Slab–> Solution –> Current –> fragments move
DNA fragments are loaded into an agarose gel slab
The slab is placed into a chamber filled with a buffer conductive liquid solution
A current is passed between wire electrodes at each end of the chamber
Because DNA fragments are negatively charged, when placed in an electric field they will be drawn to positive pole

Question 9

Describe the solution the agarose gel slab is placed in

Answer 9

Conductive buffer liquid solution

Question 10

Why are DNA fragments drawn towards the electric pole?

Answer 10

DNA fragments are negatively charged and are drawn to the positive electricity

Question 11

Explain how the agarose matrix acts as a “molecular sieve”

Answer 11

Smaller fragments can move more easily than larger ones, so smaller fragments will move farther than larger ones

Question 12

How do fragments of the same size behave in gel electrophoresis?

Answer 12

They stay together and migrate as single “bands” of DNA

Question 13

How is the 1% agarose gel created? What step is this in the overall procedure?

Answer 13

This is the first step. 0.5g agarose is added to 50mL TAE buffer in Erlenmeyer

Question 14

The mixture is brought to a boil by setting the dial to ___, and then ____ periodically. This is what number step in the procedure?

Answer 14

set dial to 7 and swirl periodically intil the agarose is dissolved
second step

Question 15

The mixture should cool for ___ minutes after removing it from the hot plate. Have instructor add ______to mixture and then, pour into gel apparatus. This is the __ step in the process

Answer 15

2 to 3 minutes
ethidium bromide
third step

Question 16

After pouring, you immediately add the ___, which is the ___ step in the procedure

Answer 16

the comb
fourth step

Question 17

While the gel soldifies, you should ___, which is the fifth step in the procedure

Answer 17

remove digested samples from the refrigerator

Question 18

For the sixth step: add ___ uL (microliters) of sample loading dye “LD” into tube, using a new tip for each sample. Close the caps on all the tubes and mix by ___

Answer 18

5uL
mix by gently flicking or tapping

Question 19

The total reaction volume after adding the enzyme mix and DNA samples is ___ uL

Answer 19

20

Question 20

In the seventh step, the casting tray should be rotated __ degrees once the gel has hardened, and the __ should be removed. The wells should be at the (?) cathode end, where the __ is connected. Carefully, remove the comb from the gel by____

Answer 20

rotate 90 degrees and remove comb
connected at (-) end where black lead is connected.
Carefully remove by pulling it straight up.

Question 21

In the eighth step, the buffer should be poured until ____

Answer 21

the buffer just covers the wells and fills both chambers

Question 22

In the ninth step, a separate tube containing __uL of ____ is obtained from instructor. ___uL of loading dye is added to this tube

Answer 22

5uL of KB ladder
5uL of loading dye is added

Question 23

The gels are read from ___ to __. The first sample is loaded in the well at the __ hand corner of the gel.

Answer 23

left to right

first sample is loaded at the left hand corner of the gel

Question 24

In the tenth step, __uL of each sample is added to the gel, with the exception of KB ladder DNA size marker which is __uL in Lane ___

Answer 24

20 uL of each sample

10uL of KB in Lane 1

Question 25

When the lid is secured in the gel box, the lid will attach in the following orientation:

Answer 25

red to red and black to black

Question 26

In the twelth step, the power supply is turned on and set to ___ V and the samples are electropheresed for ____

Answer 26

100 volts
one hour fifteen minutes

Question 27

Once the electrophoresis is complete, carefully nudge gel off tray with your __ and slide it into ____

Answer 27

with tumb
into wrigh boat

Question 28

To view the gel, look on the ___

Answer 28

transilluminator

Question 29

How are the DNA fragments measured in the quantitave analysis?

Answer 29

use a ruler to measure migration distance of each band in lane with KB ladder. Measure distance in millimeters from bottom of loading well to center of each DNA band

Question 30

The standard curve is created by using the ___ for x-axis and ___ for y-axis

Answer 30

distance X fragment Y