Insert & Vector Digestion
Restriction enzyme digestion is commonly utilized for preparation of DNA in traditional cloning
techniques to create compatible ends enabling ligation. However, restriction enzymes provides
generation of compatible ends on PCR products. For digestion of the DNA appearing in either
directional or non-directional insertion into the compatible vector, one or further restriction enzymes
are used in all cases. Restriction endonuclease also called restriction enzyme, a protein produced by
bacteria that cut the DNA at particular sites. In bacteria, these enzymes are used to cleave a foreign
DNA therefore elimination infecting organisms. Restriction endonuclease can be isolated from
bacterial cells and utilized in experimental purposes for manipulation of DNA fragments containing
the gene of interest, thus they are essential to genetic engineering. Each restriction enzymes
recognizes a specific sequence of DNA which is called restriction sites randomly distributed along
the molecule. The sugar-phosphate bonds undergo cutting reaction without damaging the bases.
(Britannica, RE)
The restriction enzymes are classified such as Type I, Type II and Type III. Type I & type III
restriction enzymes can be manipulated DNA by cutting outside of their recognition site while Type
II RE cut inside the recognition sequence which is palindromic. However, there are two types of
restriction enzymes differing in the cutting manner. Blunt end cutters cut both strand of the target
DNA at the same blot generating blunt ends whereas, sticky end cutters cut the double strand of the
target DNA at different place generating 3’ or 5’ overhangs of 1 to 4 nucleotides named as sticky
ends. For being capable of cloning a DNA insert into an expression vector, both of them have to be
treated with the restriction enzymes producing compatible ends as mentioned before. (gkp990)
Briefly in this part of the experiment, the expression vector was pEGFP-N2 isolated from E.coli and
the insert DNA was produced by PCR amplification and further gel extraction method. XhoI
from Xanthomonas holcicola (NEB) from and EcoRI from Escherichia coli (NEB) were used to cut
the full-length insert while the HindIII from Haemophilus influenza and EcoRI cut truncated version
of the insert. These selected restriction enzymes are compatible by cutting properly within Multi
Cloning Site (MCS) of pEGFP-N2 plasmid containing the restriction sites of XhoI, HindIII and
EcoRI.
The requirement of subcloning is the utilization of 1-2 restriction enzymes that cut
immediately outside the insert segment without cutting within the insert and vector themselves
therefore, the specific restriction enzymes was correctly selected from the commonly used restriction
enzymes. In this case, the gene of interest has to be inserted in the correct orientation and in frame
with the transcriptional promoter is essential.
In our experiment, the transcriptional promoter in the pEGFP-N2 vector is CMV promoter which in
frame with XhoI, HindIII and EcoRI. For full-length insert the convenient restriction enzymes were
XhoI and EcoRI which are actually done in our part.
The Polymerase Chain Reaction (PCR) is generally used to amplify a gene or DNA segment of
interest regardless of the source DNA, to be cloned. For creating compatible ends, Addition of
restriction sites to the 5’ end of the forward and the the reverse primers is common. While designing
the primers, the recommendation is to include bases between the recognition site and 5’ ends of the
primer, leading sufficient DNA for restriction enzyme to effectively bind to recognition site and cut.
The aim of this section is to digest the isolated pEGFP-N2 vector and the full-length insert DNA with
the selected restriction enzymes XhoI and EcoRI after that, visualization of the result of the restriction
digestion process was accomplished by agarose gel electrophoresis under UV illuminator. The
linearized pEGFP-N2 and the gene of our interest can be ligated though the ligation reaction in the
respect of the formation of compatible ends to each other. When the obtained recombinant pEGFPN2 will be utilized for
the further experiments such as transformation, transfection for the
investigation of the protein product belongs to the gene of our interest.
Figure 1. The Experimental structure of the insert digestion.
Figure 3. EcoRI
Restriction Site.
(NEB)
Figure 2. XhoI Restriction Site. (NEB)
Figure 4. The representation of Multi Cloning Site of pEGFP-N2 expression vector. In this
experiment, for insertion of full length of MCS protein sequence XhoI and EcoRI restriction sites are
used and the vector cut
from
these
Whereas,
sites.
for
the
insertion of truncated
version of the gene of
our interest, HindIII and
EcoRI are the restriction
sites to be cut. The
image is created by
SnapGene.
from:
Retrieved
https://www.snapgene.com/resources/plasmid-
files/?set=fluorescent_protein_genes_and_plasmids&plasmid=pEGFP-N2
Figure
5.
pEGFP-N2,
a
mammalian expression vector
represented with its origin of
replication region, promoters,
multi-cloning
site,
antibiotic
resistance genes kanamycin and
Neomycin and EFGP.
Retrieved
from
https://www.snapgene.com/resources/plasmid-
files/?set=fluorescent_protein_genes_and_plasmids&plasmid=pEGFP-N2
Gel extraction Recovery of DNA From Agarose Gel: Purification of the Insert (PCR Product)
and Digested Vector
Agarose or polyacrylamide gel electrophoresis is commonly utilized as a high resolution method to
fractionate DNA by size, is significant both as preparative and analytical procedure for isolation
individual segments of DNA molecules from a mixture. The gel extraction of the DNA is widely used
in molecular cloning experiments for downstream procedures such as restriction digestion, PCR and
sequencing. All of these requires high quality and purity of the recovered DNA. The major challenge
of the preparative use is the recovery of DNA from separate gel bands in sufficient yield and released
from gel matrix contaminants. To visualize the desired DNA fragments on the gel, ethidium bromide
is used as fluorescent staining. There are a variety of the DNA recovery techniques: Electroelution,
Free-squeeze, DNA recovery by Low Melting Point agarose gel and DNA recovery kits. Firstly,
electroelution is a simple technique by excising the the gel piece containing the gene of our interest
and placing in a dialysis bag filled with buffer and then electrophoresis results in the migration of
DNA out of the gel into the dialysis bag buffer. The purification of the DNA by performing further
steps completed. However, gel compression is the another technique to purify the DNA fragment
from the agarose gel.
First the freezing step must be taken
to make the agarose gel more
compressible. After electrophoresis, cutting the bands of interest out our the gel and equilibrating the
slices in a neutral slat buffer are performed. The freezing step must be taken to make the DNA
fragments more compressible. Furthermore, the DNA
containing buffer is squeezed though
centrifugation. Moreover, recovering DNA according to the low melting temperature of agarose gel
is also possible due to the organic-inorganic phase extraction. A several type commercial kits
including binding resins such as glass fiber matrixes, diatomaceous or silica are performed. The
mechanism is simple: The denaturation of the DNA is occurred by the chaotropic agents, leading to
bind the silica membrane.In most cases, silica membrane is negatively charged but high concentration
of caltrops makes the DNA-silica binding possible due to the fact that chaotropic salts like guadinium
thiocyanate or hydrochloride leads to break the hydrogen bonding and interrupt Van der Waals forces
between water molecules and the nucleic acids in the optimal conditions. The impurities such as salts,
free nucleotides polymerase and oligonucleotides are quickly and easily washed away after which the
elution part with sterile water or TE buffer. However, ionic salts buffer cause the decrease in the DNA
solubility by neutralizing the polynucleotide chain. Also, ethanol containing waning buffer helps the
DNA solubility to decrease by attracting the water molecules.
In our experiment, the aim of this step is for the purification of the Insert (PCR Product) and and the
digested vector (pEGFP-N2 digested with XhoI and EcoRI for the full length version and pEGFPN2 digested with HindIII and EcoRI for the truncated version of the PCR Product) by using … DNA
Recovery Kit.
REF ler AGE chrome bookmarks ta
1. Encyclopaedia Britannica. Encyclopaedia Britannica, Inc. “Restriction enzyme”. November 15th
2020. https://www.britannica.com/science/restriction-enzyme
2. Wil A. M. Loenen, David T. F. Dryden, Elisabeth A. Raleigh, Geoffrey G. Wilson, Noreen E.
Murray, Highlights of the DNA cutters: a short history of the restriction enzymes, Nucleic Acids
Research, Volume 42, Issue 1, 1 January 2014, Pages 3–19, https://doi.org/10.1093/nar/gkt990
3. International New England BioLabs. New England BioLabs, Inc. “XhoI”. November 15th 2020.
https://international.neb.com/products/r0146-xhoi#
4. International New England BioLabs. New England BioLabs, Inc. “EcoRI”. November 15th 2020.
https://international.neb.com/products/r0101-ecori#Product%20Information
5. International New England BioLabs. New England BioLabs, Inc. “Restriction Digestion
Enzyme”.
November
15th
2020.
https://international.neb.com/applications/cloning-and-
synthetic-biology/dna-preparation/restriction-enzyme-digestion
6.
Materials
• 0.2 ml Eppendorf microtubes
• 1.8ml Eppendorf microtubes
• Micropipettes (0.5-10μl), (10-100μl) and 100-1000μl) with their convenient tips
• 10X Fast Digest Buffer
• Isolated pEGFP-N2 vector
• Restriction enzymes: XhoI and EcoRI for full length version the PCR Product.
• Distilled water
• The PCR Products from the former experiment.
• Ice box
• Centrifuge
• Incubator
• 1 kb DNA Ladder
• UV illumunator
• Agarose Gel Electrophoresis apparatus
• Flask for biological waste
• Power supply
Insert & Vector Digestion Methods
1. The ingredients were kept on ice and dissolved with the help of our hand temperature.
2. Respectively, 10 µl of distilled water , 2 µl of 10X Fast Digest Buffer, 6 µl of the isolated plasmid
DNA, 1 µl of XhoI restriction enzyme, 1 µl of EcoRI restriction enzyme were placed into a 1.5ml
micro-centrifuge tube labelled as RED Vector.
3. Respectively, 5 µl of distilled water, 3 µl of 10X Fast Digest Buffer, 20 µl of the insert PCR, 1 µl
of XhoI restriction enzyme, 1 µl of EcoRI restriction enzyme were placed into a 1.5ml microcentrifuge tube labelled as RED PCR. The total volume reached at 30 µl in the RED PCR tube.
4. After the solutions were prepared, he components of the bottom of the micro-centrifuge tubes
were briefly spanned in 2 minutes.
5. The reactions were incubated at 80 °C for 5 minutes in the incubator.
6. The digested vector DNA and the digested insert DNA were loaded into the related wells.
7. Agarose gel electrophoresis was performed at 80V for 1 hour.
Table 1.The Master mixes for both the vector and the insert digestion.
Vector
Insert PCR
10X Fast Digest Buffer
2μl
3μl
DNA
6μl*
20μl
XhoI
1μl
1μl
EcoRI
1μl
1μl
dH2O
10μl
5μl
Total
20μl
30μl
* Nanodrop result for the isolated plasmid DNA concentration is 335ng/μl. The required amount of
DNA was 2μl. The calculation was the following. x was defined as desired μl.
* 335ng/μl.x μl = 2μl.1μl, x = 5.97μl approximately x = 6μl.
Gel Extraction Metarial pıçak
Gel Extraction Methods
1. Digested linearized vector and insert bands was recognized under UV light after gel
electrophoresis step.
2. Bands were appeared as orange colored under the UV light the gel image was not taken.
3. The extraction was performed by the aid of pıçak minimizing the gel of the samples
4. Two tubes were labelled as GE vector for the plasmid DNA and GE PCR for the insert DNA.
5. The labelled splendor tubes were filled with 500ul of Binding Buffer one by one.
6. Two extracted gels were placed in appropriately labelled and buffered tubes.
7. To mis the components, the tubes were vortexed.
…..
Results
Table 1. The Nano-drop results of Group 3.1 detecting by the spectrophotometer. The concentration
of the extracted vector was 28,6 ng/ul. Additionally, the 260/280 ratio of the extracted pEGFP-N2
vector was 1,83 whereas the 260/230 ratio of it was 1.47. However, the concentration of the extracted
insert DNA was 21.7 ng/ul. Furthermore, the 260/280 ratio of the extracted insert DNA was 1,84
whereas the 260/230 ratio of it was 1,16.
Restriction Digestion Results
Gel Extraction Results
Restriction Digestion Discussion
Gel Extraction Discussion
Group # 3.1
vector
PCR
concantration (ng/ul)
28.6
21.7
260/280
1.83
1.84
260/230
1.47
1.16
In this experiment, traditional restriction enzyme digestion were utilized for the formation of
compatible ends for the further step which is the ligation of plasmid vector and the PCR insert. The
pEGFP-N2 expression vector isolated from E. coli had to be linearized and include the compatible
ends with the insert DNA likewise the the gene of our interest can be digested with the same restriction
enzymes for the expression vector. The selection of restriction enzymes which are able to digest both
the vector and the insert in an convenient way was performed…