Cover page for BTEC1015 Plasmid Identification Report
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Plasmid Identification Experiment
Rufus Tolbert
Introduction
Plasmids are small, circular pieces of DNA that is capable of being replicated. Plasmids are used
to transport a gene of interest into another cell for replication, making it possible to replicate the gene
of interest into multiple copies. Restriction enzymes are catalytic enzymes that cut a DNA at restriction
sites, which are encoded in the amino acids of the DNA. The Enzymes travel over the surface of the DNA
until they come up upon their specific restriction site in the amino acid sequence. They then cut it in one
of two ways, either blunt (no overhanging amino acids) or sticky (overhanging amino acids). A single
piece of DNA can be cut once, multiple times, or not at all. It is all dependent on the presence of their
restriction site. Gel Electrophoresis is used to measure the length of pieces of DNA after being cut by
Restriction Enzymes. You place the gel in between a negative and positive charge, insuring that the DNA
fragments are closer to the negative charge because of their tendency to run to a positive charge. They
will begin to rush towards the positive charge through the gel, separating the fragments by size because
smaller fragments can travel easier than the larger pieces. Thus, the small pieces make it farther through
the gel than the larger fragments by the time you turn off the machine. Next to your plasmid, you run a
DNA ladder that is filled with fragments of very specific sizes, that way you can measure the length of
your fragments by their relation to the fragments on the ladder. The goal of this experiment was to
identify a specific, unknown, plasmid by digesting it with certain Restriction enzymes to get fragments
that eliminate the extra possible plasmids until you reach the only plasmid that has those sized
fragments with those specific Restriction Enzymes. This goal is important because it helps to understand
and put to use all of the information and skills taught in the Biotechnology 1015 class. My strategy for
identifying my unknown plasmid consists of digesting my plasmid with PVUII, BGLII, and KPNI as well as
having a lane occupied with an undigested Plasmid. This would slowly eliminate the possible plasmids
one by one leaving only one left for the unknown to be.
Methods
The code name that was given for my unknown plasmid was Br. It is possible to determine the
concentration of the Plasmid by using the spectrophotometer to get an absorbance, then calculating the
concentration of your absorbance using Beer-Lambert. The DNA ladder that was used in the experiment
was purchased from New England Bio-Labs and was product name was 1kb DNA Ladder. The Enzymes
used were also purchased at New England Bio-Labs. The names of the Enzymes were BGLII, KPNI, and
PVUII. The first Enzyme used was PVUII. The second digest used BGLII. The third and last digest consisted
of a mixture of KPNI and PVUII. The Enzymes were incubated for 9hrs at a temperature of 37 degrees
Celsius. The percentage of Agarose used in the gel was 1%. The creation of the gel involved mixing .5g of
Agarose with 50ml of 1X TAE. The mixture was then placed inside of a microwave and was melted
completely. The melted mixture was then spun in ice to cool down the temperature. It was then placed
inside of a tray with a comb consisting of 10 wells. The mixture was left to cool and solidify into the gel
that would be used. The buffer used for running the gel during electrophoresis was 1X TAE. The gel was
run at 150 volts for 30 minutes. The image of the gel was recorded using a UV Transilluminator. The
sizes of the fragments in the gel were discovered by comparing the lanes with the digests to the lane
with the ladder, lining each band up with the marker bands in the ladder symbolizing their sizes.
Predicting the sizes that would result from the digests came from an online site at:
http://tools.com/NEBcutter2/. The website had the ability to virtually digest the plasmid of your choice
with the enzyme/s of your choice. I simply digested each of the possible plasmids with the same
enzymes to discover the fragment sizes that would distinguish them from each other.
Results and Conclusions
The starting concentration of the plasmid was 17.4 micrograms per microliter. Each lane of the gel used
was a combination of 11.5ul of plasmid as well as a mixture of water and restriction enzymes to create
an overall volume of 15ul in each lane.
Gels:
pAMP
PVUII
# Ends Coordinates
Length
(bp)
1
PvuIIPvuII
2230-54
2364
2
PvuIIPvuII
375-2229
1855
3
PvuIIPvuII
55-374
320
BGLII
#
Ends
1 BglII-BglII
KPNI +PVUII
Coordinates Length (bp)
939-938
4539
# Ends Coordinates
Length
(bp)
1
PvuIIPvuII
2230-54
2364
2
PvuIIPvuII
375-2229
1855
3
PvuIIPvuII
55-374
320
pGLO
PVUII
NONE
BGlII
NONE
KPNI+PVUII
#
Ends
Coordinates Length (bp)
1 PvuII-PvuII
3052-494
2880
2 PvuII-PvuII
495-3051
2557
pBLU
PVUII
#
BGLII
NONE
KPNI+PVUII
Ends
Coordinates Length (bp)
1 PvuII-PvuII
3052-494
2880
2 PvuII-PvuII
495-3051
2557
#
Ends
Coordinates Length (bp)
1 KpnI-PvuII
3156-494
2776
2 PvuII-PvuII
495-3051
2557
3 PvuII-KpnI
3052-3155
104
pGEM
PVUII
#
Ends
Coordinates Length (bp)
1 PvuII-PvuII
2004-4821
2818
2 PvuII-PvuII
4822-2003
2113
BGLII
NONE
KPNI+PVUII
#
Ends
Coordinates Length (bp)
1 PvuII-PvuII
2004-4821
2818
2 PvuII-PvuII
4822-2003
2113
Table of Actual fragment sizes
No Digest
PVUII
BGLII
KPNI + PVUII
5500
5000
5000
5000
Table Predicted Fragment sizes
Sizes in (bp)
No Digest
PVUII
BGLII
KPNI + PVUII
pAMP
4539
2364, 1855, 320
4539
2364, 1855, 320
pGLO
5371
None
None
5371
PGEM
4931
2818, 2113
None
2818, 2113
pBLU
5437
2557, 2880
None
2776, 2557, 104
pAMP
PVUII
BGLII
pGEM
NONE
pGLO
KPNI + PVUII
NONE
NONE
pBLU
NONE
Based on the information that was attained from the gel picture as well as from the predictions, I have
come to the conclusion that the plasmid that I have, is the pGLO. pGLO is the only plasmid that still
consists of a high amount of bp’s after being digested by KPNI and PVUII at the same time. All the other
plasmids were drastically lower in the amount of bp’s.
References:
The Free Dictionary by Farlex, 2011
<http://www.thefreedictionary.com/plasmid+DNA>
New England Bio-Lab NEBcutter V2.0
http://tools.neb.com/NEBcutter2/
New England Bio-Lab Products
<http://www.neb.com/nebecomm/products/categories.asp>