Viet Pham

100266332
21/01/2017
Lab 3 DNA Restriction and Electrophoresis
1. The sample (Lambda DNA + Buffer + Water) acted as a negative control.
Water cannot cut the DNA while the restriction enzymes can. We will see the
control moving the slowest in the gel because it is longest and be able to
compare the distances between fragments and the uncut DNA.

2. Since different restriction enzymes work at varying pH conditions, a specific

buffer is optimal for 1 type of restriction enzyme. Instead of using several
different buffers for our different restriction enzymes, we can use 1
compromise restriction buffer that compromises between the optimal
conditions of each restriction enzyme.

3. One function of the loading dye is to stain the DNA so that we can see it
travelling through the gel. The loading dye also contains sucrose or glycerol
that will increase the DNAs density, allowing it the sink into the well.

4. Ethidium bromide intercalates on the phosphodiester bond (sugar-phosphate

backbone) of DNA, directly altering its structure. We did not use ethidium
bromide in this lab because it may cause changes in the DNA sequence
(mutations).

5. A. If instead of buffer, we performed the electrophoresis in water, the DNA

fragments would essentially not move. The TBE buffer provides ions to allow
the current to move, thus allowing the DNA to travel through the gel to the
direction of the current, the positive electrode. The buffer also maintains the
pH of the gel.

B. If water was used instead of TBE buffer to prepare the gel, the DNA would
diffuse in the gel and make the bands in the gel look smeared. Also because
there are fewer ions in the water than there are in the buffer, the current
within the gel would be weak, causing slow migration of DNA fragments in
the gel.

C. DNA will always migrate towards the positive electrode because DNA is
negatively charged. If the electrodes were reversed, the DNA will travel
backwards, from right to left. If the electrodes were reversed in our lab, since
the wells were put on the left side, the DNA would eventually migrate out of
the gel towards the positive electrode.

6. The gel has clearly not been run long enough; the bands are compressed and
difficult to tell apart from each other when they are close. The wells might
have been poorly formed. Perhaps the comb was removed before the gel was
completely solidified, leading to the bands looking fuzzy or wavy. In the H2O
lane, the fragment looks like its on a slant, further supporting that the gel
 was not fully set, or moved when setting. Another reason that could explain
why the bands in our gel look blurry/fuzzy is damaged wells due to pipetting
the DNA into the wells. We could have damaged the right side of the wells
(the direction of migration) leading to fuzzy/distorted looking bands. Overall,
the ideal gel looks much more crisp and clear, while our gel has a much
poorer resolution most likely due to improper pipetting, or poorly set gel.

7. A. A
casting tray that was moved when solidifying would cause impurities, such as
air bubbles, in the gel, causing some areas of the gel to be more dense and
others to be less dense. This would make the bands look wavy or fuzzy (like
our gel), or make some bands travel faster through less dense areas or
slower in more dense areas. The walls of the casting tray, for example, might
be more thicker due to the gel adhering to the sides before solidifying. This
would cause the DNA fragments to travel quicker on the edges because there
is less resistance there. A gel subject to jarring would show unreliable results.

B. Increasing the voltage of electrophoresis would increase the rate of

migration of the bands; however, increasing the voltage would also create
more imperfections in the gel. Since increasing the voltage would generate
more heat, the gel would begin to melt and change its sieving properties, and
as a result the resolution of the bands will be not as good as with a lower
voltage. Also any imperfections in the gel, small or large, would be further
notable because since the current is stronger, the bands also move faster. To
move faster they will take the path of least resistance through the gel, so a
DNA molecules path might change to get to the positive electrode quicker.
This change might cause the bands to look slanted or blurry.

C. If there were impurities in gel, like an air bubble or debris, the bands would
be altered. Because the DNA fragments travel through the gel, any impurities
in the gel would cause the bands to become distorted. For example if there
was an air bubble, the DNA would migrate quickly in that area, leading to a
band that looks bent in that area. If I use for example, | to represent a
normal band, then a small air bubble in the middle of that band would cause
it too look like >. The air bubble allows the DNA molecules there to quickly
move through the gel, because there are actually is no gel in that one area
 D. If there was too much DNA in a lane, the bands would look smeared
because there is simply too much uncut DNA that travels at a similar speed.

E. An incomplete digestion would cause very faint looking bands. Since the
enzymes cut up the DNA into fragments, an incomplete digest would lead to
more DNA being uncut and less DNA fragments migrating. Less DNA
fragments migrating would show up as less bright bands.
8.

HindIII EcoRI BamHI

Distance Bp MAP Distanc Bp Graph Bp Map Distanc Bp Bp
Migrated e e Graph Map
(mm) Migrate Migrate
d (mm) d (mm)
43 27491 44 18500 24756 48 16500 16841
45 23130 62 11000 7421 51 14750 12275
57 9416 71 8000 5804 63 10500 7233
66 6557 75 7000 4878 65 9700 6770

78 4361 88 4700 3530 70 8300 5626

108 2322

116 2027

G. The larger
fragments
were least accurate
while the smaller
fragments
were more
accurate.
This tells us that because there are larger fragments with similar sizes
relative to each other, they will appear as a single double band on the gel, and
as a result look thicker and harder to measure individual bands.
11. E. BamHI creates a fragment 12275 bp long from the left most 5505 site and
the right most 6770 site. EcoRI creates a fragment 24756 bp long using the 21226
site and the 3530 site. The fragments created are at the ends of the circular DNA.
We cannot locate these fragments individually because they appear as a doublet
with other large fragments of similar size. These together would appear as brighter
and thicker bands.