Identification of pBLU with transformation, purification, and electrophoresis using

E.coli and plasmid #10

Written By:
Walker N. Foland

LBS 145
Section Monday 7-9:50pm
Rick Chalmers and Dan Gutteridge
April 27, 2004

Abstract: by Walker Foland

The goal of the study was to investigate the identity of an unknown plasmid. To
do that, three experiments were performed. The first experiment of the study was
transformation. Transformation can be best defined as the uptake of plasmid DNA into a
bacterium. This method was used to allow the Escherichia coli to display antibiotic
resistance characteristics brought on by the gene expression of the plasmid. Three
different antibiotics were used to isolate the transformed bacteria; Ampicillin, Tetracyclin,
and Kanamycin. The second portion of the study was amplification and purification.
These processes together isolated the unknown plasmid and amplified its quantity in
order for further analysis to occur. The third and final portion of the study was DNA
fingerprinting. It consisted of two parts; restriction enzyme digest and electrophoresis. In
the restriction enzyme digest the plasmid was cut at specific base pair locations with two
enzymes. The lysed DNA were ran in an electrophoresis gel. The unknown plasmid was
found to be Ampicillin resistant in both amplification and transformation. Using a Hind
III and Ava II restriction digest, bands of DNA were observed through gel
electrophoresis. Those bands were compared to a known DNA restriction digest known as
Lambda HindIII digest. Using a logarithmic graph, the calculated base pair lengths
corresponded to the predicted base pair lengths of pBLU. Then a second electrophoresis
gel was ran with the known plasmid pBLU along with the unknown plasmid. The
unknown bands and pBLU bands matched perfectly. It was concluded that the unknown
plasmid DNA was pBLU.

Introduction: by Walker Foland

Bacteria are among one of the oldest life forms on Earth. It exsisted long before
humans or any other type of multicellular organism (Wassenaar 2003). Not only are
bacteria one of the oldest life forms on the planet, it is also one of the most parasitic.
Many diseases have risen due to bacterial infection in humans and all creatures alike.
Such a disease, which can be caused by bacterial infection, is Pneumonia.
Pneumonia is an infection of the lung tissue, which results in difficult breathing, possible
delusions, fever, shivering, coughing, and chest pain (Net Doctor 2003). It is caused by a
bacterium called streptococcus pneumoniae. This disease is seen as being quite dangerous
to the elderly. It has much more of a hold on those with weakened immune systems
resulting in a much higher rate of death. Overall bacterial infections have been a bain to
the human race. Pneumonia is currently the 7th leading cause of death in the US. In 2001
it killed 62,034 people in the United States alone (CDC 2004). Bacteria has been studied
for hundreds years, but has not been thoroughly understood or analyzed until the past 100
years. The development of antibiotics was and is still a weapon of massive importance to
the prevention of disease. However, bacteria have many methods to create resistance to
adapt to the onslaught of microscopic assassins thrown at them.
Bacteria have many dynamic characteristics, which enable them to change. One
of those characteristics, which allow them to adapt so quickly and successfully, is a
circular piece of DNA known as a plasmid (Krha et al. 2003). Plasmid DNA can be
exchanged from cell to cell in the bacterial world. Plasmids carry instructions for the
bacterial cell to develop proteins to allow a certain resistance to any number of diverse

conditions (Krha et al. 2003). Understanding how plasmids work can significantly impact
cures for bacterial infection and therefore, lower cases of disease caused by them.
Escherichia coli was used in this study to express the gene coding of the unknown
plasmid. To display such characteristics, the bacteria had to uptake the plasmid. The
process of enabling bacteria to uptake a plasmid is known as transformation.
Transformation has duel importance. One, only a small amount of DNA was given to
analyze, to go on with the planned experiments more would be needed. Two, in order to
test the plasmid for antibiotic resistance it must have a host in order to translate resistant
proteins. LB agarose plates were treated with three different antibiotics in order to grow
the transformed bacteria. The prediction was that the successfully transformed bacteria
would survive on one or more of the antibiotically treated plates. This would isolate
transformed bacteria only. Once antibiotic resistant bacteria were isolated successfully,
then amplification in Luria-Bertani (LB) broth (also treated with antibiotics) occurred.
Amplification created a large colony of bacteria and therefore synthesized more plasmid
DNA. Using three different chemical solutions the transformed and amplified bacteria
were broken down to isolate the DNA plasmid only. Then through restriction digest the
unknown plasmid was lysed at specific base pair locations to allow DNA fingerprinting.
This would enable the calculation of fragment weight for the plasmid through a line
equation from graphing a Lambda Hind III ladder digest. Through the processes of
transformation, amplification, purification, and DNA fingerprinting positive identification
of the unknown plasmid is possible.

Methods: by Walker Foland

An unknown plasmid was investigated using Escherichia coli. The first of three steps
in identifying the unknown plasmid was a process called transformation as described on
page 105 of the LBS 145 lab manual. The LBS 145 lab provided all materials for all of
the following procedures. The amount of E.coli used in the transformation procedures
were cut in half from 50 ml to 25 ml. Therefore the amount of CaCl2 used in the
procedures were also cut in half. Two controls were used for spreading the E.coli onto the
LB agarose plates. Both controls contained no antibiotic. One was spread with
transformed E.coli and was labeled con +, indicating the presence of transformed
bacteria. The other was not spread with transformed bacteria and was labeled con- to
indicate the absence of transformed E.coli. Three antibiotics were used in the process of
pouring the LB agarose plates prior to transformation. The process of pouring agarose
plates is described on page 130 of the LBS 145 lab manual. Appropriate amounts of
antibiotic were applied to certain plates. In the experiment there were eight plates in all.
Each labeled appropriately con+, con-, amp+, amp-, tet+, tet-, kan+, and kan-. Kan, amp,
and tet stand for the antibiotics, Kanamycin, Ampicillin, and Tetracyclin respectively. The
plates were all incubated at 37 C for 18 hours.
The second of the three experiments in identification of the unknown plasmid was
amplification and purification. The processes of both are explained on pages 99 through
102 of the LBS 145 lab manual. Five centrifuge tubes were used in amplification. Each
one contained 20 l of antibiotic and 2ml of LB broth. Three tubes had Ampicillin
antibiotic and the remaining two had Tetracycline and Kanamycin. After amplification,
purification began. Purification was performed with 3 trials instead of one described in

the lab book procedures. Once purification was complete a new microfuge tube was used
to mix 6 l of DNA with 2 l of loading dye. Then the mixture was centrifuged and
pipetted into an electrophoresis gel that was prepared by the LBS 145 lab TAs. The DNA
remaining was stored at 20 C.
The third and final portion in the process of identifying the unknown plasmid was
DNA fingerprinting. Prior to gel electrophoresis, a restriction enzyme digest was
performed on the DNA subject to analysis. Two trials were performed for the first
restriction digest corresponding to two lanes in gel one. A 1.5XKGB buffer
concentration presented ideal efficiency for digestion; therefore, both microfuge tubes
were filled with 5.63 l of 4X KGB buffer, 7.4 l of DNA, and 1 l of Hind III and Ava II
restriction enzymes. Those amounts were calculated with the equation V1C1=V2C2
creating a final volume of 15 l. The procedures for restriction enzyme digest are
described on page 111 of the LBS 145 lab manual. The microfuge tube was centrifuged
for 5 seconds at 12,000 RPM and then incubated in a rotating water bath at 37 C
overnight. A digest time of one hour was required for complete digestion, however
overnight digestion was performed for extra assuredness. Before electrophoresis could
begin, the gel had to be prepared. An Erlenmeyer flask was filled with 100ml of
electrophoresis buffer and .8 grams of agarose powder. The mixture was micro waved for
approximately 2.5 minutes. Once the mixture cooled to 60 C, 4 l of EtBr was added and
mixed. The procedures for pouring the agarose electrophoresis gel are on page 119 in the
LBS 145 lab manual. The DNA was mixed with a 2 l of loading dye according to the
digest volume. The DNA was loaded into the gel using a pipette. The gel was hooked up
to electrical leads and a voltage of 80-90 volts was applied. A second gel was prepared

with a second digest and all procedures were the same except a known DNA plasmid was
used along with the unknown plasmid.
Results: by Walker Foland
Transformation was performed four different times (Figure 1). On the fourth
attempt successful transformation was achieved. The first set of plates had only growth
on the LB Agar control plates with no growth on the antibiotic treated plates. The second
set of plates had similar results. The third set of plates had enormous growth on the AMP
+ , AMP , and control plates. More growth was observed on the AMP +/- plates than on
the control plates.
After the fourth attempt at transformation E.coli growth was observed on both
controls and the Ampicillin treated plate (Figure 2). One control plate (LB+) containing
LB Agar and transformed E.coli cells had thick growth with many colonies. The other
control plate (LB-) contained LB Agar and non-transformed E.coli cells. Growth was
observed to be thick and abundant, nearly exactly the same as the LB+ control. The
growth observed on the LB+ Ampicillin plate was spotted and scarce compared to the
two control plates.
Amplification and purification of the plasmid DNA from the E.coli brought forth
strong results. In amplification, bacterial growth was observed in all three of the LB/
Ampicillin centrifuge test tubes (Figure 3). No growth was observed in the Tetracycline
and Kanamycin control tubes. Many more steps toward purification finally lead to the gel
electrophoresis of the purified plasmids. DNA was found to be present in the gel test for
all three trials performed for purification (Figure 4).

In the enzyme restriction digest two restriction enzymes were used for the plasmid
DNA, Ava II and Hind III (Figure 5). The buffer used for that digest was 4XKGB buffer,
which was diluted to 1.5XKGB (Table 1). Lambda DNA was digested and cut to make
fragment sizes of 2027, 2322, 9421, 564, 6683, and 27493 (Table 2).
Electrophoresis was performed twice with the digested DNA plasmid. The first
gel ran had one control, a Lambda DNA Hind III digest. This revealed an obvious DNA
ladder for comparison against all other plasmid digests (Figure 6). Using a logarithmic
graph, general DNA molecular weight could be calculated from the Lambda Hind III
digest (Figure 7). The first gel revealed the near positive identification of pBLU (Figure
8). Expected visible digestion fragment sizes (in base pairs) of pBLU with Hind III and
Ava II digest were 1398, 1591, and 2226. The calculated fragment sizes, from the
Lambda Hind III digest, for pBLU were 2521, 1616, and, 1375 base pairs in length
(Table 3). In the second gel, pBLU was used as a known plasmid along with the unknown
plasmid, suspected to be pBLU. The known and unknown digests were shown to be
identical (Figure 9). This confirmed that pBLU was the unknown plasmid.

Discussion: by Walker Foland

The objective of this study was to be able to positively identify an unknown
plasmid. There were three large portions to the overall identification of the unknown
plasmid DNA. The first portion of the study was called transformation, which consisted
of two parts. The process of transformation involves taking bacterial cells and using a
series of chemicals and procedures to enable a DNA plasmid to be diffused. This allows
the bacterial host to translate proteins from the plasmid to acquire antibiotic resistance
and other characteristics. The second portion was called amplification and purification, it
consisted of two parts as well. The process of amplification involves growing
transformed bacteria in a bath of LB broth and antibiotic in order to increase the amount
of DNA being studied and to further support the presumed antibiotic resistance. The third
portion of the study was called DNA fingerprinting, it was made up of three parts. The
process of DNA fingerprinting involves using restriction enzymes to cut a plasmid at
specific locations so it can be differentiated from other plasmids and DNA under the
same digest in an electrophoresis gel. The hypothesis of this study was that positive
identification of the unknown plasmid DNA could be achieved through these three
portions of the study. A large amount of evidence was gathered and all them point to the
unknown plasmid being pBLU.
The first step toward transformation was pouring LB agar plates. Eight plates
were poured and all but two control plates were treated with calculated amounts of
antibiotic. Three different antibiotics were used: Tetracyclin (tet), Kanamycin (kan), and

Ampicillin (amp). The second step in transformation was preparing the E.coli bacteria.
Twenty-five ml of bacteria were used for transformation. After transformation of the
E.coli was complete, the bacteria had to be spread over all of the LB plates. Two types of
bacteria were used for spreading on the plates. One type of bacteria was transformed and
the other type of bacteria were not transformed with the unknown DNA plasmid. The
plates with transformed DNA on them were labeled with a (+) along side there
corresponding antibiotic. Those with untransformed DNA were labeled with a (-) along
side there corresponding antibiotic. All of the plates were incubated at 37 C for 18 hours.
Growth was observed on the amp+ plate and on both controls. This supports the claim
that the unknown plasmid gives the E.coli bacteria Ampicillin resistance and that
transformation was done successfully.
Transformation was performed four times before it was successful. In the first two
times no growth was observed on anything but the control plates. This was most likely
due to unsuccessful transformation of the plasmid across the bacterial membrane. The
third attempt had enormous growth on both amp+ and amp plates, along with the
controls. This was most likely due to contamination of the amp plates.
The second portion of the study to identify the plasmid is known as amplification
and purification. Amplification was performed with five centrifuge tubes all containing
LB broth. Three tubes contained Ampicillin and the remaining two contained Tetracyclin
and Kanamycin. All tubes had transformed E.coli put in them from the bacterial growth
on the amp+ plate. After an overnight incubation in a gyrating oven set at 37 C, growth
was observed in the Ampicillin treated tubes, but no growth was observed in the

Tetracylcin or Kanamycin tubes. This further supports the claim that the unknown
plasmid gives the E.coli resistance to Ampicillin.
Purification was performed in order to isolate the plasmid from the E.coli. After
breaking apart the cell membrane and precipitating the plasmid separate from the other
types of DNA from the bacteria, 6 l of plasmid DNA were mixed with 2 l of loading
dye in a separate microfuge tube. This mixture was centrifuged and ran in a gel in order
to observe the presence of plasmid DNA. Three trials were performed and all three trials
showed strong bands of plasmid DNA indicating successful purification. The DNA
remaining was put in a freezer at 20 C.
The third and most important portion of the study was DNA fingerprinting. This
consisted of two parts. The first part was the restriction enzyme digest. The restriction
enzyme digest used on the plasmid had two enzymes, Hind III and Ava II. This was a
good combination of enzymes because it cut all of the possible plasmids at different
locations making for different DNA fragment lengths. The formula V1C1=V2C2 was used
in order to calculate the correct amounts of DNA, 4xKGB buffer, and restriction enzymes
to put into a new microfuge tube for digestion. Two trials or microfuge tubes were used in
the digestion, each tube identical to the other. Once all needed chemicals were in the
microfuge tubes, they were both incubated overnight to achieve complete digestion,
however only one hour was really necessary.
The second part of DNA fingerprinting was running the gel electrophoresis. Two
gels were run. The first gel was known as gel 1 and the second was known as gel 2. The
agarose electrophoresis gel was put into a bath of electrophoresis buffer. Just enough
buffer was added onto the gel to barely cover its surface by 1mm. The plasmid DNA that

had been digested was mixed with 2-3 l of loading buffer and centrifuged. The DNA
was then pipetted into the electrophoresis gel along with a prepared Lambda Hind III
digest. The gel was run for 1 hour and 15 minutes with a voltage of 80-90 volts. The
Lambda ladder digest had known values for the bands of DNA, which enabled a log
graph to be made. The log graph was used to calculate the base pair lengths of the
unknown plasmid. The calculated base pair lengths of the plasmid were 2521, 1616, and,
1375. This data matched up very well with the predicted base pair lengths of pBLU
which were 2226, 1591, and 1398.
The first gel 1 performed was actually unsuccessful. Appropriate measurements
and calculations were performed, yet the data came out to match closest to pAMP. This
was most likely due to innaccurate measurements from a blown up polaroid of the gel. It
was also likely due to imperfections and impurities in the agarose gel combined with a
premature gel run time. The second gel was much more accurate due to a longer running
time. Measurements were also much more accurately calculated using computer software.
Measurements of the gel bands were calculated using pixels and then converted to mm.
Gel 2 was performed to confirm the suspition of the unknown plasmid being
pBLU. Gel 2 was ran with one unknown, and two knowns. One known was the Lambda
ladder, and the other was pBLU. All other procedures were identical to the first gel. The
second gel revealed identical bands for the unknown and pBLU. The hypothesis was
accepted and confirmed the prior data that the unknown plasmid was in fact pBLU.

References: by Walker Foland

American Lung Association. 2004. Pneumonia.
http://www.lungusa.org/site/pp.asp?c=dvLUK9O0E&b=35691.
Accessed 4-18-04.
CDC. 2004. Deaths/Mortality. http://www.cdc.gov/nchs/fastats/deaths.htm.
Accessed 4-21-04
Krha, Maleszewski, Wilterding, Sayed, and Luckie. 2004. LBS-145 Cell and Molecular
Biology Course Packet. 48-68
Net Doctor. 2003.Pneumonia. http://www.netdoctor.co.uk/diseases/facts/pneumonia.htm.
Accessed 4-18-04.
Wassenaar, T.M. 2003. Special feature: Bacterial Diseases in History.
http://www.bacteriamuseum.org/niches/features/diseasehistory.shtml.
Accessed 4-18-04.

Figures and Tables: written by Walker Foland

All four attempts

First Attempt

Fourth and successful attempt

Second Attempt

Third Attempt

Figure 1: Four attempts at Transformation. These are all of the plates that went into
attempting transformation. In the first and second attempts no growth was found on
anything but the control plates. The third attempt had enormous growth on the amp+/plates and the con+/- plates.

Figure 2: LB Plates. This is a picture of the LB Broth plates used in transformation.

Growth was only found on the Amp+ and control plates. The transformed E.coli cells
were therefore resistant to the antibiotic Ampicillin. Growth on the control plates were so
ubiquitous that it showed up on the plates as a thick film, not easily visible in this
photograph.

Kanamycin

Ampicillin

Tetracycline

Figure 3: Amplification Centrifuge Tubes. (Top) These are pictures of the transformed
bacterial amplification tubes for the amplification portion of the study. Each was treated
with a different antibiotic (indicated below). Growth can be seen by the white pellet in
the base of the centrifuge tube and the cloudy color of the fluid. Growth was found in the
Ampicillin tube, but not in the Kanamycin or Tetracycline tubes. (Bottom) All trials of
amplification are seen here; Three trials of amp treated tubes and one trial of tet and kan
treated tubes.

Figure 4: Presence of DNA. This is a photograph of the miniprep gel in lab 2. This
shows evidence of DNA after purification from transformed E.Coli bacteria. The bright
colors displayed are from the traveling bands of DNA gradually collecting Ethidium
Bromide (EtBr) as it travels from cathode to anode on the agarose gel. There are three
lanes due to three repetitions of the experiment to improve the probability DNA presence.

Figure 5: This is the plasmid map of pBLU, the predicted unknown plasmid yet to be
identified. The remainder of the possible plasmid maps are available in the appendix. Cut
sites are shown on each map labeled appropriately with which restriction enzyme used.
Below is a key to find the remaining maps.
Plasmid map
PUC 19
PBR322
pAMP
pKAN
pGPS3
PACYC177
Lambda

Appendix Page Number

1
2
3
4
5
6
7

Figure 6: DNA Fingerprint. This is an illustration of the expected DNA fingerprint from
Gel Electrophoresis for all seven plasmids. The far right side of the graphic shows the
length of the DNA strands post-lysis in the Lambda DNA-Hind III Digest. It is measured
in Base Pairs (BP). This is to give a reference in the digest to other plasmids and their
DNA strand lengths. Compare the pBLU drawing to the photographs of the actual gel, the
resemblance is impeccable.

Hind III ladder digest

100000
Line Equation
-2.4207

y = 5E+07x

Base Pairs

10000
1000

Hind III BP
Power (Hind III BP)

100
10
1
0

20

40

60

80

Length (mm)
Figure 7: Log graph of Lambda Hind III digest. This graph provides the basis for
calculating the molecular weight (number of base pairs) for the unknown plasmid.
Plugging in the number of mm of distance the unknown plasmid traveled through the gel
would give the approximate molecular weight of the unknown plasmid.

Figure 8: Identification of unknown plasmid DNA. This is a photograph of the first

gel electrophoresis. On the far left there is two trials of the unknown plasmid and one
trial of the Lambda Hind III digest. Comparing this photograph to that of the predicted
gel graphic in figure 5, visually the unknown plasmid can be seen as being pBLU.

Figure 9: Positive Identification of unknown plasmid. In this photograph there are two
lanes of DNA. One is the unknown plasmid and the other is the known plasmid. The
known is pBLU and perfectly mirrors the unknown plasmid. To the far right of the photo
there is a Lambda Hind III ladder.

Table 1: Restriction digest recipe. This is a table giving direction to the amounts of
DNA, Restriction Enzyme, Buffer, and dH2O that should go into digesting each one of
the plasmids. It also gives calculated digest times for each.

DNA (l )

Enzyme (l )

Enzyme 2 (l )

PAMP- 7.375 l

Hind III- 1 l

Ava II- 1 l

PUC 18/19- 7.375 l

Hind III- 1 l

Ava II- 1 l

PBR322- 7.375 l

Hind III- 1 l

Ava II- 1 l

pBLU- 7.375 l

Hind III- 1 l

Ava II- 1 l

pKAN- 7.375 l

Hind III- 1 l

Ava II- 1 l

pGPS3- 7.375 l

Hind III- 1 l

Ava II- 1 l

PACYC177- 7.375 l

Hind III- 1 l

Ava II- 1 l

Buffer (l )
4 X KGB5.625 l
4 X KGB5.625 l
4 X KGB5.625 l
4 X KGB5.625 l
4 X KGB5.625 l
4 X KGB5.625 l
4 X KGB5.625 l

DH2O

Total

Digest Time

N/A

15 l

1:00

N/A

15 l

1:00

N/A

15 l

1:00

N/A

15 l

1:00

N/A

15 l

1:00

N/A

15 l

1:00

N/A

15 l

1:00

Order

Length (BP)

From

To

1
2
3
4

3616
1798
888
222

Ava II- 3161

Ava II- 1363
Hind III- 233
Ava II- 3161

Hind III- 233

Ava II- 3161
Ava II- 1121
Ava II- 3383

1
2
3

1390
222
2506

Hind III- 447

AvaII-1837
AvaII-2059

AvaII-1837
AvaII-2059
Hind III- 447

1
2
3
4
5
6
7
8
9

1744
770
664
303
279
249
222
89
42

Ava II- 1760

Hind III- 29
Hind III- 29
Ava II- 1136
Ava II- 1481
Ava II- 887
Ava II- 3504
Ava II- 779
Ava II- 1439

Ava II- 3504

Ava II- 799
Ava II- 3726
Ava II- 1439
Ava II- 1760
Ava II- 1136
Ava II- 3726
Ava II- 887
Ava II- 1481

1
2
3
4

2226
1591
1398
222

Ava II- 1599

Ava II- 1599
Hind III- 3190
Ava II- 4588

Ava II- 4810

Hind III- 3190
Ava II- 4588
Ava II- 4810

1
2
3
4
5
6

1390
1000
814
748
222
20

Ava II- 3038

Hind III- 234
Ava II- 1234
Ava II- 2068
Ava II- 2816
Ava II- 2048

Hind III- 234

Ava II- 1234
Ava II- 2048
Ava II- 2816
Ava II- 3038
Ava II- 2068

1
2
3
4
5
6
7
8

1264
950
661
510
434
222
155
42

Ava II- 139

Hind III- 3385
Ava II- 1625
Ava II- 2286
Ava II- 2951
Ava II- 1403
Ava II- 2796
Ava II- 97

Ava II- 1403

Ava II- 97
Ava II- 2286
Ava II- 2796
Hind III- 3385
Ava II- 1625
Ava II- 2951
Ava II- 139

pAMP

pUC 19

pBR322

pBLU

pKAN

pGPS3

pACYC177

1
2
3
4
5
Lambda/ Hind III
1
2
3
4
5
6

1303
1215
766
435
222

Ava II- 403

Ava II- 2907
Ava II- 1706
Hind III- 2472
Ava II- 181

Ava II- 1706

Ava II- 181
Hind III- 2472
Ava II- 2907
Ava II- 403

27493
9421
6683
2322
2027
564

Hind III- 44149

Hind III- 27481
Hind III- 37466
Hind III- 25159
Hind III- 23132
Hind III- 36902

Hind III- 23132

Hind III- 36902
Hind III- 44149
Hind III- 27481
Hind III- 25159
Hind III- 37466

Table 2: Fragment sizes. This shows the entire set of fragment sizes acquired from the
restriction enzyme digest of all of the plasmids. The two right columns describe the cut
locations of plasmid by the restriction digest and which enzymes are doing the cutting.
The column labeled length is the base pair lengths of DNA resulting in the restriction
digest.

Table 3: Calculated molecular weight of unknown plasmid. This table shows the
distance traveled by the unknown plasmid fragments (left column) along with the
corresponding molecular weight (center column). Molecular weight was calculated using
the formula y = 5E+07x-2.4207 (right column). The calculations come very close to the
predicated molecular weight of pBLU. BP stands for base pairs.
Band travel of
Calculated
Unknown Plasmid (mm) Unknown Plasmid BP
59.6
2521.339824
71.62
1616.175184
76.56
1375.202158

Predicted
pBLU BP
2226
1591
1398

Appendix