Lab Chapter 4: Part Two Flashcards
Describe the PPE required for this week’s lab
Nitrile gloves
What does the 3D structure of restriction enzymes allow them to do?
attach to a double stranded DNA molecule and slide along until they recognize a sequence of base pairs.
What will a restriction enzyme do if there is multiple restriction sites in a DNA molecule?
It will cut the DNA molecule into multiple fragments
What determines the length of each fragment?
depends on the location of restriction sites in DNA molecule
What does agarose gel elctrophoresis do?
When restriction enzymes cut multiple fragments, they can be separated and visualized with this process.
What does the term electrophoresis mean?
to carry with electricity
How does electrophoresis separate DNA fragments?
According to their relative size
Describe general process of electrophoresis
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
Describe the solution the agarose gel slab is placed in
Conductive buffer liquid solution
Why are DNA fragments drawn towards the electric pole?
DNA fragments are negatively charged and are drawn to the positive electricity
Explain how the agarose matrix acts as a “molecular sieve”
Smaller fragments can move more easily than larger ones, so smaller fragments will move farther than larger ones
How do fragments of the same size behave in gel electrophoresis?
They stay together and migrate as single “bands” of DNA
How is the 1% agarose gel created? What step is this in the overall procedure?
This is the first step. 0.5g agarose is added to 50mL TAE buffer in Erlenmeyer
The mixture is brought to a boil by setting the dial to ___, and then ____ periodically. This is what number step in the procedure?
set dial to 7 and swirl periodically intil the agarose is dissolved
second step
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
2 to 3 minutes
ethidium bromide
third step
After pouring, you immediately add the ___, which is the ___ step in the procedure
the comb
fourth step
While the gel soldifies, you should ___, which is the fifth step in the procedure
remove digested samples from the refrigerator
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 ___
5uL
mix by gently flicking or tapping
The total reaction volume after adding the enzyme mix and DNA samples is ___ uL
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____
rotate 90 degrees and remove comb
connected at (-) end where black lead is connected.
Carefully remove by pulling it straight up.
In the eighth step, the buffer should be poured until ____
the buffer just covers the wells and fills both chambers
In the ninth step, a separate tube containing __uL of ____ is obtained from instructor. ___uL of loading dye is added to this tube
5uL of KB ladder
5uL of loading dye is added
The gels are read from ___ to __. The first sample is loaded in the well at the __ hand corner of the gel.
left to right
first sample is loaded at the left hand corner of the gel
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 ___
20 uL of each sample
10uL of KB in Lane 1
When the lid is secured in the gel box, the lid will attach in the following orientation:
red to red and black to black
In the twelth step, the power supply is turned on and set to ___ V and the samples are electropheresed for ____
100 volts
one hour fifteen minutes
Once the electrophoresis is complete, carefully nudge gel off tray with your __ and slide it into ____
with tumb
into wrigh boat
To view the gel, look on the ___
transilluminator
How are the DNA fragments measured in the quantitave analysis?
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
The standard curve is created by using the ___ for x-axis and ___ for y-axis
distance X fragment Y
