Lateesha Thomas 620099129

Course: Molecular Biology II

Code: BIOC3312
Date: September 12, 2018
Name: Lateesha Thomas
Title: Analysing the size of isolated Recombinant Plasmids by rapid miniprep procedure
Aim:
 Determining biochemically that the transformed cells contained plasmid DNA
 Determining that the size of the DNA is consistent with the expected size
 Determining the size of the DNA using gel electrophoresis

Abstract:
In this experiment, size analysis of recombinant plasmid was done using gel electrophoresis and
rapid miniprep procedure to determine that transformed cells contain plasmid DNA. Miniprep
procedure was used to suspend pellets of samples in different reagents such as EDTA, glucose
and Tris- HCL and was vortex mixed. The same reagents where added again with lysozyme and
the sample equilibrate at room temperature. Other reagents where added and the tubes inverted
and place on ice for a while and centrifuged. The supernatant was removed and transfer into
another tube, reagent added, it was centrifuged, supernatant removed, and pellets allowed to dry.
Agarose gel was prepared using TBE buffer and wells were formed. Different reagents where
added to the tubes with pellets to adjust the volume and loading gel was added to each tube and
the sample loaded in the wells and electrode connect and it was left to electrophorese overnight.
It was found that the transformed cells contained plasmid DNA this was detected due to the
comparison of size of the DNA using the electrophoresis since the size of the plasmid is known.
The size that was known for the plasmid is 4176. This procedure was found to be an effective
way to determine the size of the DNA to show that the transformed cells contained plasmid
DNA. The expected size was consistent with the DNA size
Lateesha Thomas 620099129

Introduction:
Agarose gel electrophoresis can be the easiest and commonest way of separating and analysing
DNA fragment based on the size. The purpose of the gel might be to look at the DNA, to
quantify it or to isolate a band. The DNA sample to be used are added to the wells and a voltage
added which allow the negatively charge DNA to move towards the positive end of the gel. This
movement is based on the different size and conformation of the DNA fragment with each band
movement. The small pores of the agarose gel act as a sieve that provides great power. Small
molecules move quickly through the pores than larger molecules. Large molecules have more
resistance as they make their way through the pores and therefore travel slower. The different
size and net charge are factors that determine how quickly molecules will travel through the
agarose gel. Small size and strong charge increase a molecule’s migration rate through the gel.
Large size and weak charge decrease the migration rate. The DNA is visualised in the gel by
addition of ethidium bromide, which is mutagenic, or less-toxic proprietary dyes. The DNA
bands take up this dye as they move along the gel. Ultra violet light allows this dye to appear
pale pink in colour. The larger fragments appear brighter under the light since they take up more
dye than the smaller fragments.
In this experiment a rapid miniprep procedure was used. This procedure involves the extraction
of plasmid DNA from bacterial cell suspensions and in this case to determine if the transformed
cells contain the plasmid DNA. The fact that plasmids are small supercoiled DNA and bacterial
chromosomal DNA is larger and less supercoiled is taken advantage of in this procedure. Cells
are lysed under alkaline conditions which denatures proteins and nucleic acids. The solution is
then neutralized with Ammonium acetate, proteins precipitate along with chromosomal DNA
because they cannot renature correctly since they are large. However, plasmid renature and stay
within the solution which allow for the separation from the proteins and chromosomal DNA.
In the miniprep procedure the cells of the sample are suspended in Tris- HCL, glucose and
EDTA and vortex mixed. The Tris- HCL is used to maintain a stable pH within the sample along
with the help of glucose. EDTA however, helps to prevent the DNA from being disrupted which
increases its yield, this is done by EDTA binding to Mg2+ ions and depriving nucleases of this
co-factor. Lysozyme is also added to the mix since it helps to improve the efficiency of the DNA
extraction, but it is used mostly to lyse bacterial cells. The sample is equilibrated and a solution
of NaOH and SDS is added. The purpose of NaOH is for lysing, it is an alkaline lysis buffer that
is used to dissolve cell membrane. This allow for the DNA to come out. On the other hand, SDS
(sodium dodecyl sulfate) is a detergent that is used in DNA isolation to solubilize the proteins
and lipid from the membrane. After NaOH and SDS are added the sample is equilibrate on ice
then Ammonium acetate is added. This function as a mean to precipitate proteins, high molecular
weight RNA and bacterial chromosomal DNA. The sample is then centrifuged, clear supernatant
removed and isopropanol added to the sample. Isopropanol function as a reagent that precipitate
DNA out of the solution the concentration of isopropanol caused the DNA to become insoluble.
Since DNA molecules are soluble in water but not soluble in organic solvents. However ethanol
that is used to wash the DNA pellet, function by changing the potential of DNA to ionic to
remove water molecules this aid in the precipitation of the DNA. It
Lateesha Thomas 620099129

Method:
Experiments in Molecular Biology: Biochemical Applications by Zachary Burton and Jon
Kaguni.
Pages: 137- 139
Modification made:
Lateesha Thomas 620099129

Results:
Agarose gel Electrophoresis showing the separation of the pTrc99A/ TopA DNA that was
stained with ethidium bromide, loaded on the gel and separated using 30 V (constant voltage)
along with the control and DNA ladder.

Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample
Position Position Position Position Position Position Position Position Position Position
1kb B1 B2 B3 B4 B5 B6 +ve
_ DNA Control _
LAdder
Lateesha Thomas 620099129

Discussion:
In this experiment the size analysis of recombinant plasmid was done using the miniprep
procedure. With this procedure the plasmid DNA was isolated and ran on agarose gel to observe
the DNA typological forms and to find out if the plasmid DNA took up the recombinant DNA
The DNA sample that was used were added to the wells of the electrophoresis apparatus and a
voltage of 110v was added for one and a half hours for the DNA to run along the gel. That is the
negative DNA moves toward the positive end of the gel. This movement occurs based on the size
since supercoiled DNA moves faster than linear and nicked DNA. The different size and net
charge are factors that determine how quickly molecules will travel through the agarose gel.
Small size and strong charge increase a molecule’s migration rate through the gel. Large size and
weak charge decrease the migration rate. The DNA is visualised in the gel by addition of
ethidium bromide, which is mutagenic, or less-toxic proprietary dyes.
On the other hand, after the agarose gel electrophoresis was done, the gel photograph was
observed to see if the recombinant plasmid size was consistent with the expected size. On the
results eight wells where observed with the first being the 1kb DNA ladder and the last one the
positive control. In all wells coming down there were bands seen that have been separated
showing the different typological forms of the DNA. The one that is the closest to the positive
charge on the electrophoresis can be consider to be supercoiled DNA since it is supercoiled and
easily passes through the gel. Following supercoiled you can find linear DNA that travels a bit
slower than supercoiled then after the linear you have nicked DNA. This consist of a plasmid
DNA with a break on one of the strands. These bands were also observed in the positive control
which indicate that the plasmid took up the recombinant DNA.
However, even though it was observed that the bands from the different wells were in line with
the positive control, the bands however were above the 1kb DNA ladder. This was not expected
since it was expected to find the bands within the range of 6.0 to 8.0 kb for the complete
recombinant sample DNA since it consist of Ptrc99A that was known to be 4176bps and the
inserted TopA cys B gene which is known to be 3819bps.

Limitation:
Questions:
Reference (APA):