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Experiment 2: Spectrophotometric Measurement of DNA, Digestion
of Vector and Insert DNA, Agarose Gel Electrophoresis, and Gel
Purification
INTRODUCTION
Restriction endonucleases are enzymes typically produced and used by bacteria as a host defense
mechanism [1]. They catalyze the sequence-specific recognition and cleavage of double stranded
DNA and have been used extensively in practically all recombinant DNA applications [2]. Three
different types of restriction enzymes have been identified with Type II being the most
commonly used one, due to the fact that it performs the cleavage directly within or in close
proximity to the sequence it recognizes unlike the other two types of endonucleases [2, 3].
Endonucleases can either produce sticky ends with 3′ or 5′ overhangs or blunt ends with no
protruding nucleotides [3]. Complementary sticky ends can hybridize giving the ligation process
unidirectionality whereas with blunt ends, reversions may occur [3].
After a restriction reaction is completed, the desired DNA fragments are isolated typically by
performing agarose gel electrophoresis on the digested products, then excision of agarose slices
containing DNA which are visible under UV light thanks to EtBr which can bind to the DNA by
intercalating between base-pairs from either of the DNA grooves but more strongly bound when
from the minor groove [4, 5]. The desired fragments can be recovered using various methods
such as using commercial kits containing binding resins such as silica or glass fiber matrixes;
freeze and thaw-squeeze of the DNA-containing agarose; the use of low melting agarose and
DNA release into solution by heating; or electroelution with utilization of dialysis bags and
electric current [6].
To extract DNA from agarose gel using commercial purification kits, chaotropic salts are used to
denature the DNA so it can bind to the resin inside the spin column [6]. Wash buffers with
ethanol are used to cleanse the DNA from impurities such as restriction enzymes and agarose,
followed by removal of ethanol and the elution of DNA using distilled water [6].
After purification of DNA, the nucleic acid concentration should be measured to be able to
calculate the amount of elute that should be used in further experiments as well as to determine
DNA purity and the presence of contaminants [7]. The most common method for this purpose is
nanodrop spectrophotometer in which the max absorbance wavelength of DNA which is 260 nm
is used to quantify the concentration and 260/280, 260/230 absorbance ratios which are ideally
1.8 and 2.0 – 2.2 respectively, are used to determine contamination by protein and or phenol, and
unwanted organic compounds such as guanidine thiocyanate respectively [7,8].
AIM
In this experiment we aim to perform endonuclease digestion on the insert DNA which was
previously amplified in PCR and pEGFP-N2 vector in its relevant MCS by using the staggered-
end producing restriction endonucleases HindIII and EcoRI, then performing gel electrophoresis
on the obtained digestion products to purify the linearized vector and staggered ended insert
DNA using a commercial gel purification kit to obtain two pure DNA samples with
complementary staggered ends so that they can be ligated to produce a circular plasmid in future
experiments and be used in further studies such as bacterial transformation. The purity and the
concentration of purified digested DNA will be determined using nanodrop spectrophotometer.
MATERIALS & METHODS
Materials: • 5.7 μL of 350 ng/μL pEGFP-N2 vector, • 20 μL Insert PCR Product, • 5.5 μL 10X
enzyme buffer containing dye, glycerol and EtBr, • 2 μL HindIII endonuclease, • 2 μL EcoRI
endonuclease, • distilled water, • 0.8% agarose gel, • UV transilluminator, • scalpel, • weighing
scale, • GeneAll® ExpinTM Gel SV (102-150, 102-102) (Column, Buffer GB, Buffer NW), •
microcentrifuge, • 0.8% agarose gel, • gel tray, • power supply, • TAE buffer, • thermal
incubator, • Eppendorf microtubes, • nanodrop spectrophotometer, • micropipettes, • tips
All materials were kept on ice while preparing the digestion tubes.
14.8 μL of
dH 2
O
, 2.5 μL of 10x enzyme buffer, 5.7 μL of vector DNA, 1 μL of EcoRI, 1 μL of
HindIII were mixed in a tube by adding in the respective order, the tube was labeled as vector
DNA. (See appendix)
RESULTS
pEGFP-N2 vector and the PCR product were subjected to restriction digestion using EcoRI and
HindIII restriction endonucleases to produce linear DNA fragments with complementary sticky
ends. The digestion products were loaded and run on agarose gel electrophoresis. The resulting
bands were excised and gel purification using GeneAll® ExpinTM Gel SV (102-150, 102-102)
commercial kit were performed. The purified DNA was analyzed using a spectrophotometer.
The recorded values for vector digestion-gel purification product were:
Concentration: 10.3 ng/μL
The recorded values for PCR product digestion-gel purification product were:
Concentration: 5.3 ng/μL
DISCUSSION
Our goal was to perform endonuclease restriction reaction on pEGFP-N2 vector and the PCR
product to produce complementary staggered ended linear DNA fragments that can be ligated
unidirectionally. To eliminate contaminants such as endonucleases and the cut oligonucleotide
from the vector and ensure that the restriction reactions were successful we performed gel
electrophoresis and gel purification using a commercial gel purification kit. The purified DNA
samples would then be analyzed in nanodrop spectrophotometer to determine the purity and
concentration.
Our gel electrophoresis results were as expected with single linear specific bands for both the
vector and the PCR product. The oligonucleotide was not visible on the gel because, due to its
extremely short length it would migrate out the gel very quickly. The band lengths were as
expected which are about 4.7 kb for the vector and 300 b for insert DNA hence the usage of 4f
primer in previous PCR experiment.
The concentration values of 10.3 ng/μL and 5.3 ng/μL implicate the success in gel purification
for both the PCR product and the vector DNA. The 260/280 ratios of 1.93 and 1.99 are
sufficiently close to the ideal value of 1.8 and imply no phenol or protein contamination and
because the numbers are not larger than 2.0 maybe a mild basic contamination [8]. The 260/
ratios of 0.87 and 0.41 are much smaller than the acceptable range of 2.0 - 2.2. This strongly
suggests contamination with a contaminant absorbing at 230 nm or less. A larger value would
mean organic contamination such as with as guanidine thiocyanate [8]. This contaminant may
have been incorporated into the products during the wash step in gel purification and further
centrifugation steps may have failed to eliminate the contamination. Purity-wise vector product
seems to be better due to the larger 260/230 value possibly due to the high beginning
concentration of 360 ng/μL.
APPENDIX
