Rosemarie Dawn Tagare

Experiment 2: DNA Isolation, Restriction Digest, and Electrophoresis

January 27, 2015
Lab Partner: Mary Prior
TA: Yoonjung Shim

Abstract:
The purpose of this experiment was to explore recombinant DNA technology and to
understand how bacterial plasmid DNA is prepared, purified, cleaved by restriction enzymes, and
visualized using gel electrophoresis. Understanding central physiological processes such as
protein-nucleic acid and nucleic-nucleic acid interactions is important for developing an
understanding and identification of various diseases. DNA purification was carried through a
series of buffer additions followed by centrifugation. The restriction enzyme HindIII was used to
cut the circular plasmid at four recognition sites, which would create four segments of DNA. The
absorbance of two dilutions (200x and 100x) of the isolated DNA was taken at 260 nm and 280
nm to determine the concentration. The absorbance at 260 nm was 0.142 and 0.222 for 200x and
100x DNA solutions, respectively. This corresponded to concentrations of 1.42

200x DNA sample and 1.11

g
L

g
L

for the

for the 100x DNA sample. The 260/280 ratio of the

absorbances was calculated to be 1.42 and 1.22, and the disparity signified an impure sample.
However, ultimately the experiment was successful because 4 bands were found around ~4200,
~3400, ~1000 and ~900 bp during gel electrophoresis. The bands (4252 bp, 3423 bp, 1036 bp
and 928 bp were close enough to the expected values to understand which ones corresponded to
each other.
Introduction:
Recombinant DNA technology has become revolutionary in biomedical research. In this
experiment, we aimed to use this technology to purify a pVL 1392/1393 plasmid from E. coli
cells, cleave it with HindIII restriction enzyme, and then perform gel electrophoresis to visualize
the DNA bands. Recombinant DNA is a form of artificial DNA that is covalently ligated in
juxtaposition to heterologous DNA sequences that is propagated into an appropriate host1; in our
case, the host is E. coli. A series of buffers were used to isolate and purify the plasmid DNA. P1
is a resuspension buffer containing RNase A. RNase A is a pancreatic bovine ribonuclease that
destroys single-stranded RNA molecules at cytosine and uracil residues2. P2 buffer was an
alkaline lysis buffer containing NaOH. It degraded the DNA membrane and denatured protein

and chromosomal DNA. N3 buffer containing acetic acid and guanidine-HCl was used to
neutralize the basic solution and renature plasmid DNA. The chaotropic salt in this buffer causes
cellular debris to precipitate. PB, which contained isoproanol and chaotropic salt, allowed
binding between the DNA and Si-based column. Lastly, PE was a washing buffer containing 7080% ethanol that removed the remaining salt. After purification of the plasmid DNA, the
absorbance was taken for the sample using a quartz cuvette and spectrophotometer. A high purity
DNA sample usually has the ratio

Absorbance at 260 nm
Absorbance at 280 nm

between 1.65 and 1.85. We could

calculate the concentration of the DNA from the absorbance using the formula:

DNA concentration = Absorbance Units

50

g
doublestranded DNA
mL
1 Absorbance Unit

Dilution Factor.
After determining the miniprep DNA concentration, a restriction endonuclease (REase)
was used to cleave the remaining sample of DNA at a specific recognition site. These enzymes
cut the DNA within the molecule. Restriction enzymes are integral in the formation of
recombinant DNA. The REase used in this experiment was HindIII. Its name is derived from the
genus and species from which the enzyme was isolated (Haemophilus influenzae), followed by
letters and numbers inactive of the individual strains or serotypes (third strain, serotype d)3.
HindIII cleaves the DNA whenever it encounters the palindromic sequence
5 A A G C T T 3
3 T T C G A A 5
between the adjacent adenines. After cleaving, the overhanging single-stranded DNA fragments,
the sticky ends, can complementary base pair with other molecules. The double-stranded DNA
can then be joined by the enzyme DNA ligase, which will result in recombinant DNA.
Gel electrophoresis was performed to separate the fragments of DNA cleaved by HindIII.
Before loading the samples, an ethidium bromide dye was added in order to visualize them under

UV light. After loading the samples and ladder into the wells of the gel, the nucleic acids are
separated by size due to the negative charges on the DNAs phosphate backbone. When an
electric field is applied throughout the agarose gel, the negatively-charged fragments will migrate
toward the positively-charged anode. The shorter fragments are found near the bottom of the gel,
since they can travel faster than the larger ones4.
The vector used in this experiment was pVL 1392/1393, which has 9369 base pairs. Its
four HindIII restriction sites are found at 1 bp, 4253 bp, 5181 bp, and 6217 bp, which resulted in
bands of lengths 4252 bp, 3243 bp, 1036 bp, and 928 bp. These bands can be visualized by
viewing the gel under a UV light. In

order to estimate the fragments

lengths, we compared the band

positions to those of a 1-kbp marker.

Figure 1 shows the standard DNA

ladder which can be used to compare

the observed DNA samples to

determine its length.

Figure 1. Sample DNA 1-kbp ladder.

By comparing locations of the bands in our gel to the ones we predicted, we could
determine the success of our experiment.
Materials and Methods:

Materials:

Eppendorf pipettes

Eppendorf tube

(P1000, P200, and P20)

P1 Buffer with RNase A

Disposable tips of

N3 Buffer

QIAprep column

appropriate sizes for

PB Buffer

Quartz Cuvette

Buffer 2

USB-650 Red Tide

Distilled H2O

BSA

Ethidium Bromide

each of the pipettes

E. Coli cell pellet

Hind III

Microfuge tubes

Eppendorf Centrifuge
5424

Spectrometer

1% Agarose gel

Loading Dye

Plasmid Preparation:

An E. Coli cell pellet in a was obtained from the TA and resuspended in 250 L of

P1 buffer. The cell suspension was transferred to a 1.5-mL microfuge tube. Next, 250 L of P2
buffer was added, which lysed the cells. After inverting the closed tube five times, 350 L of N3
(neutralizing) buffer was added. Once again, the tube was inverted five times, then spun down in
the centrifuge for 10 minutes at 14000 rpm. While centrifuging, a QIAprep column was placed in
2-mL collection tube. Since the supernatant exceeded the capacity of the column, half of the it
was transferred to the column using a pipette, and spun down for one minute. The flow-through
was discarded, and the remaining supernatant was transferred to the column and spun down for
another minute. After discarding the flow-through, 500 L of PB (binding) was added to the
column and centrifuged for one minute. The flow-through was discarded, then 750 L of PE
(washing) buffer was added to the tube and centrifuged for one minute. After discarding flow
through, the empty tube was spun down for an additional minute in order to remove residual
washing buffer. The spin column was removed and transferred to a 1.5 mL micro-centrifuge tube.
30 L of deionized water was used to elute the DNA. The column was allowed to stand for one
minute before centrifuging for one minute. The resulting solution was collected.

DNA Determination:
In order to determine the DNA concentration, we created two dilutions from the
sample. For a 200x dilution, 497.5 L of distlled water and 2.5 L of the purified DNA
were placed in a microfuge tube. In another tube, 495 L of distilled water and 5.0 L of
the puried DNA were added in order to have a 100x dilution. The spectrometer was
zeroed using a blank (distilled water). Then, 10 L of the 200x diluted sample was
pipetted into a quartz cuvette and the absorbance was taken using the USB-650 Red Tide
spectrometer. The absorbance values were recorded at wavelengths of 260 nm and 280
nm. This was repeated for the 100x diluted sample. The information was compiled in
Table 1.

Restriction Digest:

The concentration of DNA was determined using the recorded absorbance values
at 260 nm. Based on this concentration, we elected to use 3.0 L of the purified DNA in
order to have around 3 g. This meant that 40.5 L of distilled water had to be added for
the restriction digest. The other components necessary for the digest included: 0.5 L
100x BSA, 5 L of 10x Buffer 2, and 1 L of HindIII enzyme, which brought the total
volume to 50 L. When all contents were added to the Eppendorf tube and mixed gently,
the sample was incubated for eight hours at 37oC. Our TA removed the tubes from the
incubator and transferred them to a freezer where they were stored until we ran gel
electrophoresis.

Gel Electrophoresis:

A pre-made 1% gel was placed into the chamber of the gel electrophoresis

machine. The gel was created by dissolving 2 g agarose powder in 200 mL 1x TAE buffer, and
heated. Then 10 L of ethidium bromide was added. The mixture was poured into a mold, and a
comb was placed near the top in order to form wells. The cooled gel was stored until usage in gel
electrophoresis. The frozen restriction digest sample was thawed then placed on ice. 10 L of 6x
loading buffer was added to the tube in order to visualize the bands. The TA added 30 L of the
1-kbp marker in the first well, then 30 L of the sample was loaded into the next well of the gel.
The gel was run for 40 minutes at 150 Volts. Once complete, the gel was viewed under a UV
lamp. A picture was taken for reference (Figure 2).

Results:
Table 1. Absorbance data was retrieved from the two diluted solutions of purified plasmid DNA.

D
i
l
u
t
i
o
n
2
0
0
x
1
0
0
x

2
6

Composi
tion

Absorbance at
260 nm

Absorbance at
280 nm

/
A
2
8
0

497.5 L
ddH2O +
2.5 L
DNA
495 L
ddH2O +
5.0 L
DNA

0.142

0.134

0.222

0.173

Based on the table above, the DNA concentration was calculated. The following
calculations were performed.

For 100x dilution:

0.142 Absorbance Units

500 L distilled water + DNA

5 L DNA

0.222 Absorbance Units

500 L distilled water + DNA

2.5 L DNA

= 1110

g
mL
1 Absorbance Unit
50

g
mL

1 mL
1000 L

= 1.11

g
L

For 200x dilution:

1 mL
1000 L

= 1420

= 1.42

g
mL

g
mL
1 Absorbance Unit
50

g
L

The volume of the original sample needed for 3 g DNA was calculated from the
concentrations.

1
.
0
6
1
.
2
8

100x: 3 g

1 L
1.11 g

= 2.70 L

200x: 3 g

1 L
1.42 g

= 2.11 L

Our TA advised us to round this number to 3.0 L. Since the total volume of the

mixture had to equal 50 L, the amount of distilled water used = 50 L (3.0 L of miniprep
DNA + 0.5 L of 100x BSA + 5.0 L of 10x Buffer 2 + 1.0 L of HindIII enzyme) = 40.5 L of
ddH2O.

Figure 2. The DNA sample after it was run through the agarose gel and the 1-kbp marker for
comparison.

Discussion:

The purpose of this experiment was to explore recombinant DNA technology and

to understand how bacterial plasmid DNA is prepared, purified, cleaved by restriction enzymes,
and visualized using gel electrophoresis. The absorbance at 260 nm was found to be 0.142 and
0.222. Since the difference in the dilution factor was 2x, the absorbance values should have also

differed by 2x. The DNA is considered pure if the ratio is about 1.8. However, since the ratios for
both diluted samples (1.06 for 200x and 1.28 for 100x) were less than this, this meant there were
contaminants such as protein or phenol that have strong absorbances around 280 nm. The
concentration of DNA was found to be 1.11

200x dilution. The

Absorbance at 260 nm
Absorbance at 280 nm

g
L

for the 100x dilution and 1.42

g
L

for the

ratios and the concentration values should have

been equal for the 100x and 200x samples of DNA, since the only thing that differed was the
dilution factor. The impurities that have caused these disparities may have resulted from several
errors. For example, a significant amount of pipetting was utilized in this experiment. In the
previous lab, we showed that the Eppendorf pipettes were not very accurate, which may have
affected our results. By cleaving the DNA with HindII, 4 bands should have been observed after
gel electrophoresis at bands of lengths 4252 bp, 3243 bp, 1036 bp, and 928 bp. Our bands
appeared orange under the UV light. By comparing them to the 1-kbp ladder, we determined the
locations of our bands to be at ~4200, ~3400, ~1000 and ~900 bp.
Overall, our experiment was successful since we were able to distinguish 4 separate
bands corresponding to the ones we predicted to see on the gel. The discrepancies in absorbance
ratios and concentration may have been minimized if we had more accurate piptettes and a
meticulous pipetting technique. Resolution of bands could have been greater if the gel was run
for a longer period of time.

References:

1. Gardner, D. G., & Baxter, J. D. (1982). Recombinant DNA. The American Journal of
Medicine, 72(4), 551-553. doi:10.1016/0002-9343(82)90443-0
2. RNase A, DNase and Protease-free. http://www.thermoscientificbio.com/dna-and-rnamodifying-enzymes/rnase-a-dnase-and-protease-free.
3. Nucl. Acids Res. (2003) 31 (7):1805-1812.doi: 10.1093/nar/gkg274
4. Koontz, L. (2013). Agarose gel electrophoresis., 529 35-45. doi:10.1016/B978-0-12418687-3.00004-5

Appendix

Please refer to attached notebook pages.