Gel electrophoresis lab

Victoria Liu
Purpose
The objective of this experiment are one, it aims understand the use of gel electrophoresis to
separate DNA based on their size and charge. Two, it allows the experimenters to practice
scientific technique of inserting DNA into wells using pipets.

Materials
- Plasmid DNA (Uncut)
- Plasmid cut with bgl I
- Plasmid cut with Eco RI
- Lambda DNA (uncut)
- Lambda DNA cut with Eco RI
- Lambda DNA cut with bgl I
- UltraSpec-Agarose ™
- Electrophoresis buffer (50x)
- Practice Gel Loading solution
- FlashBlue ™ DNA Stain
- InstaStain ® Blue cards
- 1 ml pipet
- Microtipped Transfer pipets
- Horizontal gel electrophoresis apparatus
- D.C. power supply
- Automatic micropipets with tips
- Balance
- Hot plate
- Pipet pump
- 250 ml flask
- Hot gloves
- Small plastic trays (for gel destaining)
- Distilled water

Introduction
Restriction enzyme led to a new age in molecular genetics. These endonucleases
catalyze the cleavage of the phosphodiester bonds, which connect the 2 strands of DNA
(Edvotek, 1989). They require MG+2 for activity and produce 5 prime and 3 prime hydroxyl
group at end of cleavage (Edvotek, 1989). Restriction enzyme are obtained from bacteria.
They are named by the bacteria, from which they are first isolated from (Edvotek, 1989). It
consists of the first letter of the genus followed by the first two letters of the species
(Edvotek, 1989). The Roman numeral indicates the strain or sub-strain of a spieces that
produced the restriction enzyme (Edvotek, 1989). Restriction enzymes can cut DNA
sequence at a precise length of 4-8 base pairs, known as recognition sites (Edvotek, 1989).
The recognition sites are symmetrical; their base sequence are identical from direction of 5’-
3’, called palindromes (Edvotek, 1989). There are a variety of cleavage pattern in recognition
sites. The staggered cleavage of Eco RI is known to have a sticky- ends, where the single
stranded regions are complementary to each other (Edvotek, 1989). When the cuts are
opposite from each other, the cleavage is called blunt ends (Edvotek, 1989). Recognition sites
with variable base positions are known to be degenerate. Lastly, recognition sites that divide
by a particular total number of bases are known to be hyphenated sites (Edvotek, 1989). The
longer the DNA molecule is, the greater the probability of the presence of recognition sites
(Edvotek, 1989). Human chromosomes DNA with three billion base pairs will have
significantly more recognition sites than plasmid DNA with only few thousand base pairs
(Edvotek, 1989). When plasmid or circular prokaryotic chromosome have one recognition
site, the strand will become linear (Edvotek, 1989). In contrast, when linear chromosome
with one recognition site, it will separate into two. Furthermore, when DNA molecule has
several recognition site, some are incompletely cleaved, which are called partials (Edvotek,
1989). Agarose gel electrophoresis is a method to analyze DNA fragments produced by
restriction enzymes. The agarose gel has microscopic pores, which serves as molecular sieve
(Edvotek, 1989). When DNA are loaded into gel, its strong negative charge at neutral pH
makes it migrate towards positive electrode (Edvotek, 1989). The molecules are separated
according to their size and charge (Edvotek, 1989). The larger DNA molecule stay closer to
the wheel. Circular DNA are supercoiled, which has a more compact and entangled shape
(Edvotek, 1989). When supercoiled DNA is cut by restrictive enzyme, it unravels to linear
form. In electrophoresis, supercoiled DNA move faster than linear form (Edvotek, 1989). The
replication forms catenanes, which consist of dimer or trimer of plasmid molecules (Edvotek,
1989). They move closer in electrophoresis than single molecules. In this experiment, It is
expected that the same restriction enzyme can yield diverse patterns when digesting different
DNAs (Edvotek, 1989).

Data table

The effect of restriction enzyme on DNA fragment’s migration distance in gel electrophoresis

Well Total number of bands Distance migrated (cm) ± 0.5

A 2 0.8
1.2
B 0 -
C 1 0.9
D 0 -
E 1 1.5
F 1 1.6

Sketch
Analysis
Gel electrophoresis separate charged molecule in an electric field, based on their
charge and size. All the negatively charged DNA molecule move in the same positive
direction during electrophoresis, however not at the same rate. Smaller fragments move
towards the opposite direction faster than the larger ones, which allows gel electrophoresis to
separate DNA fragments according to their size. Based on the data collected, the first band in
well A and well C are the longest with the heaviest mass, as the distance migrated were only
0.8 and 0.9cm. It is followed by the DNA fragment from well the second band in well A,
which migrated 1.2 cm. Following this, the second smallest DNA fragment is 1.5cm apart
from well E. The smallest DNA is from well F, which migrated the furthest of 1.6cm.
In the sketch, the DNA fragment located 1.2cm apart from well A is extremely light,
in comparison to the dark bands present in well E or F. This is due to the varying amount of
DNA inserted into the well. The greater the mass of the inserted DNA is, the more intensely
the band will stain on the gel.
The uncut plasmid DNA and Lambda DNA are expected to have 2-3 bands, as they
are able to form supercoiled (compact and entangled shape), non-supercoiled (linear, nicked
and relaxed) and interlocking structures (Edvotek, 1989). Based on the gel electrophoresis
generated, well A has a supercoiled form followed by a circular, single strand, which is
thinner and more faint. However, well D has no DNA bars present, which is different from
the expected result. The supercoiled plasmid DNA contains 4500 to pairs have one
recognition site for Bgl I and two for Eco RI (Edvotek, 1989). In comparison to the data
collected, there was no recognition site present for BgI I and one recognition site present for
Eco RI. The Lambda DNA is expected to contain 5 recognition sites for Eco RI and 29 for
Bgl I. However, there was only 1 recognition site for present for each in the data collected.
The difference between the expected and experimental data may be caused by the
following errors:
1) The agarose gel was kept out of the fridge for 3-5 days, which makes it less
effective in showing the recognition sites.
2) The gel was ordered in September, therefore it may have expired and lost its
effectiveness.
3) The staining and de-staining technique is also a potential error source. The stain
used may have an inappropriate concentration, which results in poor visualization of the
recognition site. During the de-staining process, the constant exchange of water may have
excessively removed the stain. As a result, the DNA fragments are unable to appear clearly
on the gel.
4) During the process of transporting the gel to the fridge, it was ripped. This is a
significant experimental error that can affect the DNA frament’s visuliazation.
5) The technique of inserting DNA into the well is a potential error source, where the
pipet was not accurately placed into the well. As a result, the DNA may have flown under the
gel. This cause a lack of DNA in the well, which results in fewer DNA fragments appearing
on the gel.

Conclusion
This experiment determined the different numbers of DNA fragments created by
various restriction enzymes and met the goal of practicing the scientific technique of inserting
DNA into the gel using pipet. It also demonstrates that the same restriction enzyme can
produce various patterns, as lambda DNA cut by BGl I is expected to have 29 recognition
sites, whereas plasmid DNA cut by BGl I is only expected to have 1 recognition sites
(Edvotek, 1989). However, the significant differences between the expected and experimental
data are due to the errors that were present.

Bibliography
Restriction enzyme cleavage of plasmid and Lambda DNA. (1989). Retrieved
December 12, 2019, from https://www.edvotek.com/site/pdf/102.pdf.