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Introduction
Polymerase chain reaction (PCR), can be used to amplify rare specific DNA sequences
into many billions of molecules when the ends of the sequence are known. The method of
amplifying rare sequences from a mixture has numerous applications in basic research,
human genetics testing, and forensics. In one application, small samples of DNA, such as
those found in a strand of hair at a crime scene, can produce sufficient copies to carry out
forensic tests. In PCR Genomic DNA is digested into large fragments using a restriction
enzyme and then is heat-denatured into single strands. Two synthetic oligonucleotides
complementary to the 3' ends of the target DNA segment of interest are added in great
excess to the denatured DNA, and the temperature is lowered to 50-60o C. The genomic
DNA remains denatured, because the complementary strands are at too low a
concentration to encounter each other during the period of incubation, but the specific
oligonucleotides, which are at a very high concentration, hybridize with their
complementary sequences in the genomic DNA. The hybridized oligonucleotides then
serve as primers for DNA chain synthesis, which begins upon addition of a supply of
deoxynucleotides and a temperature-resistant DNA polymerase such as that from
Thermus aquaticus (a bacterium that lives in hot springs). The Taq polymerase can
extend the primers at temperatures up to 72oC. When synthesis is complete, the whole
mixture is heated further (to 95oC) to melt the newly formed DNA duplexes. When the
temperature is lowered again, another round of synthesis takes place because excess
primer is still present. Repeated cycles of synthesis (cooling) and melting (heating)
quickly amplify the sequence of interest. At each round, the number of copies of the
sequence between the primer sites is doubled; therefore, the desired sequence increases
exponentially (2, 16).
Polymerase Chain Reaction (PCR)
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Polymerase Chain Reaction (PCR)
The elegant technique of PCR, by which fragments of DNA can be made to replicate very
rapidly, is illustrated (3). A medical application of PCR is early detection of infection
with HIV, the virus that causes acquired immunodeficiency syndrome (AIDS). The PCR
is so sensitive that it can detect HIV at very early stages in the disease (before symptoms
appear) when only a few thousand blood cells in a patient are infected with the virus (13).
Cloning and Recombinant Expression
The discovery of two types of enzymes provided the means of the now common
technique of DNA cloning. One type, called restriction enzymes, cut the DNA from any
organism at specific sequences of a few nucleotides, generating a reproducible set of
fragments. The other type, called DNA ligases, can insert DNA restriction fragments into
replicating DNA molecules producing recombinant DNA. The recombinant DNA
molecules then can be introduced into appropriate cells, most often bacterial cells; all the
descendants from a single such cell, called a clone, carry the same recombinant DNA
molecule. Once a clone of cells bearing a desired segment of DNA is isolated, unlimited
quantities of this DNA can be prepared. The essence of cell chemistry is to isolate a
particular cellular component and then analyze its chemical structure and activity. In the
case of DNA, this is feasible for relatively short molecules such as the genomes of small
viruses. But genomes of even the simplest cells are much too large to directly analyze in
detail at the molecular level. This obstacle to obtaining pure DNA samples from large
genomes has been overcome by recombinant DNA technology. With these methods
virtually any gene can be purified, its sequence determined, and the functional regions of
the sequence explored by altering it in planned ways and reintroducing the DNA into
cells and into whole organisms. The essence of recombinant DNA technology is the
preparation of large numbers of identical DNA molecules. A DNA fragment of interest is
linked through standard 3'-5' phosphodiester bonds to a vector DNA molecule which can
replicate when introduced into a host cell. When a single recombinant DnA molecule,
composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the
inserted DNA is reproduced along with the vector, producing large numbers of
recombinant DNA molecules that include the fragment of DNA originally linked to the
vector. Two types of vectors are most commonly used: Escherichia coli plasmid vectors
and bacteriophage lambda vectors. Plasmid vectors replicate along their host cells, while
lambda vectors replicate as lytic viruses, killing the host cell and packaging the DNA into
virions (2, 14).
In the early 1970s, biochemists at Stanford University showed that genetic traits could
indeed be transferred from one organism to another. In this experiment, the DNA of one
microorganism recombined with the inserted DNA sequence of another, and thus had
been edited to exhibit a very specific modification. The actual editing, or insertion
process, is painstaking, for it involves manipulating incredibly tiny pieces of incredibly
tiny organisms. But the process can be explained in terms of editing a written text:
scissors and "glue" are used to "cut" and "paste." The methods used in rDNA technology
are fairly simple. We take, for example, the sentence (gene) for insulin production in
humans and paste it into the DNA of E.coli, the bacterium that inhabits the human
digestive tract. The bacterial cells divide very rapidly making billions of copies of
themselves, and each bacterium carries in its DNA a faithful replica of the gene for
insulin production. Each new E. coli cell has inherited the human insulin gene sentence.
To transfer the gene embodying the instruction for insulin production, it is necessary to
cut the appropriate gene from human DNA and paste, or splice, it into plasmid DNA, a
special kind of DNA that takes a circular form and can be used as a vehicle for this
editing job. Our "scissors" are the class of enzymes called restriction enzymes. There are
well over a hundred restriction enzymes, each cutting in a very precise way a specific
base sequence of the DNA molecule. With these scissors used singly or in various
combinations, the segment of the human DNA molecule that specifies insulin production
can be isolated (6, 15).
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This segment is "glued" into place using an enzyme called DNA ligase. The result is an
edited, or recombinant, DNA molecule. When this recombinant plasmid DNA is inserted
into E. coli, the cell will be able to process the instructions to assemble the amino acids
for insulin production. More importantly, the new instructions are passed along to the
next generation of E. coli cells in the process known as gene cloning (6, 19).
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(4)
Prokaryotic Expression Vectors
Yeast and other similar cells are ideal for such a purpose due to the fact that these cells
contain all of the enzymes necessary for normal eukaryotic transcription. Furthermore,
recombinant DNA molecules permit integration into the genomes of larger, multicellular
organisms such as mammals. A possible result of such an organism would be the
production of a transgenic organism--an organism in which a transformed parent (of
recombinant DNA molecules) produces the transgenic offspring, carrying integrated
foreign DNA from the parent. Expression vectors allow eukaryotic gene products to be
detected in prokaryotes. Bacterial expression vectors are usually plasmids with strong
promoters, ribosome-binding sites, and transcription terminators. Eukaryotic proteins are
made by inserting cDNA into an expression vector. Also, antibodies can be used to
screen clones from expression-vector cDNA libraries in order to identify unknown genes.
Expression of Proteins in Eukaryotes:
Since prokaryotes may still not be able to produce eukaryotic proteins (due to a
glycosylation requirement), some eukaryotic expression vectors have been established.
Yeast and other similar cells are ideal for such a purpose due to the fact that these cells
contain all of the enzymes necessary for normal eukaryotic transcription. Furthermore,
recombinant DNA molecules are able to be integrated into the genomes of larger,
multicellular organisms such as mammals.A possible result of such an organism would
be the production of a transgenic organism--an organism in which a transformed parent
(of recombinant DNA molecules) produces the transgenic offspring, carrying integrated
foreign DNA from the parent (7).
Genomic and cDNA libraries can be screened for the properties of a specific protein
encoded in the cloned DNA. This approach uses special cloning vectors, called lambda
expression vectors, in which the cloned DNA is transcribed into mRNA, which in turn is
translated into the encoded protein. For example, lambda phage vectors have been
constructed so that the junction of inserted DNA lies in a region of the vector that is
transcribed and translated at a high rate. Cloned DNA inserted at this position is
transcribed into mRNA in every cell infected by this type of vector. If the cloned DNA
contains a protein-coding sequence inserted in the same reading frame as the vector
protein, infected cells will produce a fusion protein in which the amino terminus is
encoded by the vector DNA and the remainder of the molecular by the cloned DNA.
In expression cloning, any molecule that binds to a protein of interest with high affinity
and specificity can be labeled and used as a probe to identify clones expressing the
interacting protein (2, 20).
Affinity Purification
Once a specific DNA clone has been isolated, the cloned DNA is separated from the
vector DNA by cleavage with the restriction enzyme used to form the recombinant
plasmid. The cloned DNA and vector DNA then are separated by gel electrophoresis, a
powerful method for separation proteins according to size. Gel electrophoresis also is
sued to separate DNA and RNA molecules by size and to estimate the size of nucleic acid
molecules of unknown length by comparison with the migration of molecules of known
length. DNA and RNA molecules are highly charged near neutral pH because the
phosphate group in each nucleotide contributes one negative charge. As a result, DNA
and RNA molecules move toward the positive electrode during gel electrophoresis.
Smaller molecules move through the gel matrix more readily than larger molecules, so
that molecules of different length, such as restriction fragments, separate. Because the gel
matrix restricts random diffusion of the molecules, molecules of different length separate
into "bands" whose width equals that of the well into which the original DNA mixture
was placed. The resolving power of gel electrophoresis is so great that single stranded
DNA molecules up to about 500 nucleotides long can be separated if they differ in length
by only 1 nucleotide. DNA molecules composed of up to 2000 molecules are separated
electrophoretically using polyacrylamide gels, and molecules from 500 nucleotides to 20
kb on agarose gels. Two methods are common for visualizing separated bands on a gel.
One is to incubate the gel in a solution containing the fluorescent dye ethidium, a planar
molecules that binds to DNA by intercalating between the base pairs. Binding
concentrated ethidium in the DNA and also its intrinsic fluorescence. As a result, when
the gel is illuminated with ultraviolet light, the regions of the gel containing Dna
fluoresces much more brightly than the regions of the gel without DNA. Affinity
purification, specifically, affinity chromatography, relies on the ability of a protein to
bind specifically to another molecule. Columns are packed with beads to which are
covalently attached ligand molecules that bind to the protein of interest. Ligands can be
enzyme substrates or other small molecules that bind to specific proteins. In a widely
used form of this technique, affinity chromatography, the attached ligand is specific for
the desired protein. An affinity column will retain only the proteins that bind the ligand
attached to the beads; the remaining proteins, regardless of their charge or mass, will pass
through the column without binding to it. The proteins bound to the affinity column are
then eluted by adding an excess of ligand or by changing the salt concentration or pH.
Obviously, the ability of this technique to separate particular proteins depends on the
selection of appropriate ligands (2,17). For example, in a mixture of proteins, a certain
protein we want to purify has a His Taq, or essentially a series of Histidines, attached to
the amino or carboxyl terminus. The column used is chelated and activated with nickel
(Ni+). Our protein of interest sticks to the column. The column is then eluted with
Histidine whose iminizole group will serve to elute the protein (21).
The Fluorophore of Green Fluorescent Protein (GFP)
GFP is a fluorescent protein isolated from coelenterates, such as the Pacific jellyfish,
Aequoria victoria, or from the sea pansy, Renilla reniformis. Its role is to transduce the
blue chemiluminescence of the protein aequorin into green fluorescent light by energy
transfer. The gene for GFP has been isolated and has become a useful tool for making
chimeric proteins of GFP linked to other proteins where it functions as a fluorescent
protein tag. GFP tolerates N- and C-terminal fusion to a broad variety of proteins. It has
been expressed in bacteria, yeast, slime mold, plants, drosophila, zebrafish, and in
mammalian cells. As a noninvasive fluorescent marker in living cells, it allows for a wide
range of applications where it may function as a cell lineage tracer, reporter of gene
expression, or as a measure of protein-protein interactions (12). One application of the
technology involves visualization of Microtubule (MT) dynamics in living plant cells
using the green fluorescent protein. Higher plant cells MTs show dynamic structural
changes during cell cycle progression and play significant roles in cell morphogenesis.
The cortical MT (CMT), preprophase band (PPB), and phragmoplast, all of which are
plant-specific MT structures, can be observed during interphase, from the late G2 phase
to prophase, and from anaphase to telophase, respectively. The CMT controls cell shape,
either irreversibly or reversibly, by orientating cellulose microfibril deposition in the cell
wall; the PPB is involved in determining the site of division; and the phragmoplast forms
the cell plate at cytokinesis. The appearance and disappearance of these MT structures
during the cell cycle have been extensively studied by immunofluorescence microscopy
using highly synchronized tobacco BY-2 cells. These studies that were conducted using
green fluorescent protein have revealed much about the modes of MT structural
organization and dynamic changes during the cell cycle (10). Another application
involves the human heart Na+ channel (hH1) protein, in which recent evidence suggests
that biosynthesis hH1 protein is rapidly modulated by sympathetic interventions.
However, data regarding the intracellular processing of hH1 in vivo are lacking. This
study sought to establish a model that would allow the study of the subcellular
localization of hH1 protein. Such a model could eventually help in better understanding
the trafficking of hH1 in vivo and its potential role in cardiac conduction. The C-terminus
of hH1 was labeled with the green fluorescent protein (GFP) and the expression of this
construct (hH1-GFP) was compared to the expression of hH1 in transfected HEK293
cells. The green fluorescent protein was used to show that the ER may serve as a
reservoir for the cardiac Na+ channels and that the transport from the ER to the Golgi
apparatus is among the rate-limiting steps for sarcolemmal expression of Na+ channels
(11) .
Materials and Methods
The Polymerase Chain Reaction
Stock50 ml reactions (Final Volume)
10 x Buffer 5 ml
2.5 mM dNTP4 ml200 microM
.5 ml Polymerase5 ml.5 ml
10 ng/ml Target5 ml50 ng
19 pmol/mm 5' primer5 ml50 pmol
10 pmol/ml 3'primer5 ml50 pmol
25 mM MgCl23 ml1.5 mM
Water
The above concentrations were prepared for The Polymerase Chain Reaction.
Electrophoretic Gel - Purification and Extraction
20 ml of 0.7% Agarose EtBr
0.15grams of agaroseIsopropanol
WaterElectrophoresis Setup
EDTA, TAE, Tris, and Acetic acid.
20 ml of 0.7% Agarose (Low Melt - 70oC ). Microwave 0.15grams of agarose in 20ml of
water for about 30 seconds until dissolved. Low Melt contains EDTA, TAE, Tris, and
Acetic acid.
Add 10 ml of excess EtBr (150mg/ml always).
Run HIND III Marker, 1KB DNA ladder which has about 1000, 2000, 3000 bp on it.
Add 6x loading dye - about 10©l to the tubes. Don't worry about contamination.
Apply electric field and run electrophoresis gel.
Visualize gel under UV light.
Cut out band - purified sample. Put different buffers and melt at 50oC and pull DNA
down. Get the gene out of agarose. Melt agarose in 50oC water bath. Gel pieces are
melted so liquid is left in tube. Agarose comes right out. DNA in TAE buffer.
Isopropanol is used to precipitate. Create mixture of 140ul of Isopropanol, 20ml NaAc (.3
M), and 40ul of product + water. Total = 200 ul.
DNA will pellet - clear pellet. Dump it out. Place clear pellet in 70% ethanol to get rid of
residual salt. Suck out extra ethanol using vacuum suction.
Restriction
Digest. 20 ©l digest of:10x buffer - 2ml100x BSA ®(diluted to) 10x - .2 mL ®(diluted
to) 2ulBam HI and Ecor1 - 1©l eachwater - 14 mlLB Brotheppendorf tubes and pipettes
We have .25 mg/ml we're cutting 1 microgram. Add 4ml of vector - 2ml of Bam HI and
EcoR1, respectively, 2ml of buffer, 2ml BSA, 10ml water.
Bam HI and EcoR1 - steam bath at 80oC for 20 minutes.
Restriction Digest 37oC for 1 hour. Then water bath to kill enzyme.
Place in an eppendorf tube: 1ml Ligase, 10ml Insert, 1ml Vector, 2ml 10x Buffer, 6ml
H2O.
(Cloning Rxn) Rxn Tube: 5ml Vector + Insert, 1ml ligase, 2ml buffer, 12 ml H20.
Transform E.Coli - 3ml of ligation mix ® 3ml Rxn ligation 1ml control PUC19.
30ml Competent Cells ® 3ml Rxn ligation ® 1ml control PUC 19.
Placed in ice in a Styrofoam cooler.
Ligation Rxns:3ml JM109 E.coli (cloning strain) in ice for 20 minsThen steam bath 42oC
for 90 seconds (Heat Shock)Room Temperature for 2 minutes37oC in 1 ml Luria Bertani
Broth for 30 minutes
Luria Bertani consists of: 10gl/L Tryptone - pancreatic
digest, 5g/L yeast extract, 5g/L NaCl, 15g/L Agar, 100mg/ml Ampicillin.
PlatingdishesloopBunsen burnerControl" RXN mixtures.
1100 ml each on separate plates - Control. In one plate spread the RXN mixture, in the
next plate, spread the Control, in the last place, spread both.
Ligation
Rxn Centrifuge MachinePipettestubeResuspension SolutionwaterTrisBeefer
Reagentcuvette
Spin cells down. Resuspend cells on smaller volume.100 ml Resuspension Solution contains RNAse100 ml lysis Solution - NaOH - 2 min200 ml Neutralization Buffer - Add
Salt Buffer
Resuspend digest in 50 ml H20Dave's Clone: 2 ml Buffer D, 2 ml BSA (Dilute 100x to
10x), 1 ml NdeI, 1ml xhoI, 14 ml plasmid solution.
Other Clones 2 ml multicore buffer, 2 ml BSA, 1 ml BamHI, 1 ml EcoRI, 14 ml plasmid
Solution
0.14 g of agarose gel + 20 ml of TBE, 5 ml of dye to each tube.
1 M stock Tris ph 7, 15 ml 20 mM Tris 300 ml - 2 Test Tubes. Resuspend pellets in 5ml
of 20 mM Tris.
50 ml worth of culture
Resuspend in 5 ml of Tris. Add 100 ml to cuvette +
900 ml of water so OD = to original OD.
Eppendorf 100ml of cells + 900ml of Beefer reagent
Put in 20mM of Tris - bursts outer membrane.
Add lysozyme - breaks peptidoglycan layer down. Then sonicate or French press to break
rest open.
Affinity Purificationresin imidazoleTrisWatereppendorf tubespipettes
Incubate for 20 minutes at room temperature and break open. - 1st step of purification.
To isolate target protein - subject it to hypotonic solution. Bursts. Now you have soluble
protein in solution - spin down the e.coli.
Two tubes - 500 mM imidazole in both, 20 mM Tris, 20mM + 5mM imidazole
200 ml of 20mM Tris to each! Bring up to 10 ml using water.
10ml 1 20mM Tris pH 7
10 ml 2 20 mM + 5mM 100
10 ml 3 20mM + 20mM 400
10ml 4 20mM + 100 mM 2 ml
1ml of resin + 14 ml of H20. Spin it.
Invert tube, dump soluble contents into resin tube. Add 12 ml of 20 mM Tris.
Take out 500 ml into eppendorf and pour out rest.
Resin spin down.
Aliquot the supernatant into an eppendorf.
Put 5 ml of pellet into another eppendorf.
Put 1 ml imidazole into resin. 10% SDS. 30% Starting Concentration.
Electrophoretic Gel
Resolving Gel (10%)10 mlStacking Gel (6%)(Lower) Tris pH 8.82.5 2.5 pH 6.8
(Upper)10% SDS100 ml100 mlcis:bis 29% Acrylamide3.32 mlAPS 500 ml500
mlH2O3.65.9TEMED10 ml10 ml
Run SDS PAGE. Separate proteins based on size (21).
Results and Discussion
Cloning a gene is an involved process relying not only on molecular techniques, but also
on classical genetics, protein biochemistry, and microbiology. The initial stages of
cloning are straightforward and involve:
1) isolation of DNA from the source organism;
2) fragmentation of the DNA into relatively small pieces;
3) combining the fragments with a vector (a DNA molecule which replicates in a host) to
form a recombinant DNA molecule;
4) introducing the recombinant DNA into a host;
5) screening hosts for a specific fragment of DNA (1).
We ran the PCR. Next we collected our PCR product and ran a gel. We ran HINDI III,
which is the marker. It is a lambda digest. Lambda phage produce small genomes and
fragments. We also ran a 1KB DNA ladder, which contained about 1000, 2000, 3000
base pairs on it. The purpose of the first gel we was to purify and get rid of impurities in
the DNA. DNA is negatively charged so it runs to the anode. We take our restriction
enzyme and cut it at both ends. We are purifying the DNA because there are some things
we don't want: the polymerase, the primers, and the dNTPs. The restriction enzyme will
get "confused" with the presence of the dNTPs. We don't want any more primer because
it will supply more substrate for the restriction enzyme to work with. We don't want the
polymerase because we cut our restriction enzyme. With polymerase and dNTPs, it will
extend from 5' to 3', which is not the purpose. We run a gel instead of doing a phenol
extraction to avoid nonspecific product. So we cut out the band after 716 base pairs and
put it into different buffers. We melted at 50oC and precipitated the DNA. It is important
to run a low melt agarose, which is however expensive. To visualize products, a high
melt agarose is required. For the loading dye, glycerol or Ficoll are needed and both serve
the same purpose. They provide a viscous medium for DNA. The solution does not
prevent diffusion, however. It slows down the rate of diffusion so we can load our gels.
The dye is needed to see how long the gel needs to run. The elements of creating a
plasmid include:
1) getting our PCR product
2) conducting a restriction digest
3) and sticking the plasmid into a vector.
To Design a Cloning Vector, we need a(n):
1) origin of Replication, which determines the copy number. The copy number is the
number of copies of the plasmid that exist on average within an E. coli cell. The average
copy number ranges from 50 to 80.
2) gene, selective marker, is always in the lower left hand corner of the map. The
resistance gene needs a promoter and a terminator.
We visualized the gel under UV light and collected our purified sample by cutting out the
bands. We then need to retrieve the gene from the agarose. We placed the eppendorf tube
containing the agarose piece with the gene of interest in a 50oC water bath, which melts
the agarose. Only the liquid is left in the tube and the agarose comes right out. The DNA
is left in TAE buffer and Isopropanol was then used to precipitate the DNA. Then we
spun down the tube for 15 minutes at 18,000 rct (relative centrifugal force). The DNA
then became a clear pellet. We dumped out the liquid. We put the pellet in 70% ethanol
to rid it of residual salt and rid the extra ethanol by using a vacuum suction apparatus. For
the restriction digest, we used pNEB193 Polylinker. Bovine Serum Albumin (BSA) must
be added with Bam HI to reduce star activity (when the enzyme cleaves outside its
recognition sequence). We then conducted a restriction digest and cut only 1 microgram
of the gene. The vector was then added. The Bam HI and the EcoRI was then heat
inactivated for 20 minutes at 80oC. The restriction digest was conducted at 37oC for 1
hour, and then placed in the water bath to kill the enzyme. In the ligation process, a small
piece of the vector is cut using alkaline phosphates, which takes the 5' phosphate off of
DNMA so that it cannot ligate to itself. Enzymes will then repair the DNA.
_________________
__________________
The gene of interest is 716 base pairs long.
20 BP long
Bam HI
________
________
Eco RI
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ProductsE. Coli Growth
1) ===== PCR product (undigested)O
2) © Vector product (undigested)+
3) © + © Eco RIO
4) © + © Bam HIO
5) © Eco RI+
6) © Bam HI+
7) © + ©+
8) © + © + ©O
We need 10x more inset than vector. The linear plasmid is better than the circular
plasmid. The ideal ration of insert to vector is 5:1, but here, we used 10x ml insert:
vector. We then transformed E. Coli. We got single colonies, selected them, grew them,
cut with Bam HI and Eco RI and saw if we got colonies with insert. Every lane should
have a plasmid. The two clones in by 1000 are positive clones.
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In the transformation of E.Coli, we placed the ligation mix and the competent cells in a
Styrofoam cooler. Competent cells reflect their competency to take up DNA. We increase
their competency by exposing them to CaCl or Calcium Phosphate. We placed the
ligation reactions containing JM 109 E.Coli (cloning strain) on ice for 20 minutes and
then heat shocked them for 90 seconds and then placed them in Luria Bertani Broth at
37oC for 30 minutes. 1/1000 or 1/10,000 E.Coli take up a plasmid temporarily. We
spread the solution on a petri dish. The E.Coli cloning strains do not produce T7RNA
polymerase. Some Cloning strains include XL -1 Blue, DH-S a, and JM Series. The
Expression Strains include BL21DE3. Protein expression hurts the E. Coli cell. In the
cloning vector, there is no promoter, so no protein product will be produced. Since the
T7RNA polymerase is not produced by E.Coli, we put the gene into the plasmid and
clone it. The gene product, however, will not be produced. So the basic difference
between the cloning and expression strains is that the former has no promoter so no
protein product forms, and in the latter, a protein product does indeed form. For example,
lactose repressor sits on the promoter, so in the presence of the lactose, the repressor
inactivates. Instead of lactose, IPTG an analog of lactose that is membrane permeable to
the E.Coli cell is used. In conducting the plating, 1100 ml of each tube was spread on
separate dishes; control on one, reaction tube on another, and a mix of both on the last.
For the ligation reaction tube, the cells were spun down and resuspended in a smaller
volume. We did not get any colonies. Perhaps the E.Coli did not transform correctly. The
class, however, got 1000s of clones. However, it's possible, we did not get any positive
clones. One reason the class got 1000s of clones may be due to the fact that we may not
have digested the plasmid correctly. In cloning, it is necessary to carry out the following
steps correctly:
1) Prepare Target and vector
2) Design Primers
3) PCR
4) Clean PCR Product - we are not 100% certain we got PCR product.
5) Restriction Digest - there is no way to know if the product was cut properly. We
assume it was.
6) Clean the Restriction Digest - we heat killed it. However, we are not certain whether
this worked either.
7) Ligation
8) Transformation - we know with 100% certainty that this worked.
9) Inoculation (plating)
10) Plasmid Prep
11) Restriction Digest
12) Frozen Stock
In lysing the bacterial cell wall, NaOH was used. The alkaline solution breaks down the
cell wall. The neutralization buffer contained salt buffer as well, which promotes
hydrophobic interactions so DNA binds to beads. We used the Crude Prep and
isopropanol was used to precipitate the DNA and, recentrifugation was done. 70%
ethanol was added to wash away NaCl which promotes hydrophobic and electrostatic
interactions, and hydrogen bonds. The low concentration of NaCl affects star activity;
stringency is lowered.The restriction digest was then performed with Dave's Clones and
Other Clones. The electrophoretic gel was then run; a high melt was done since we did
not excise the bands. We just wanted to look at them. The RNA is usually among the
lowest bands because most RNA is transcript. In alkaline lyses, the RNA is subject to
base catalyzed hydrolysis. We then each received different fluorophores. My group
received GFP. We then took 50 ml worth of culture, spun it down, resuspended it in 5 ml
of Tris. We then added100 ml to a cuvette and water to bring it up to 1ml so the OD = to
the original OD. The OD around 600nm was 1.965. We then put the culture in 20 mM of
Tris, which bursts the outer membrane. The inner membrane has peptidoglyan. So we
added lysozyme, which breaks down the peptidoglycan layer. Then, usually, sonication
or French press is needed to break the rest open. We then incubated for 20 minutes at
room temperature and allowed lyses to separate the soluble versus the insoluble. Soluble
GFP has HisTaq attached. If we had free Ni+, then the Histidines chelate around Nickel.
It is unlikely we had Histamines; this allowed us to make a resin. We spun down 1 ml of
resin and 14 ml of H2O. The Nickel chelates resin; it's fool proof. We added imidazole to
compete with the protein trying to bind with the resin. So then we washed it with Tris.
The tubes were then inverted and we dumped out the soluble contents into the resin tube.
We took 500 ml into a tube and spun down the resin. The supernatant was the aliquoted
into an eppendorf and we put 5ml of the pellet into another tube. We put 1ml of
imidazole into the resin and spun it down. We took out the supernatant into another tube
and once again added 1ml of imidazole to the resin. So we basically were spinning and
retrieving product at different concentrations of imidazole. We then ran an SDS PAGE.
The proteins are sandwiched between the Cl- and the glycine. SDS mask the charge
properties of proteins so we separated solely on the basis of size. We got fragments that
represent pieces of the entire genome. Then we clone into a plasmid to receive 1000s of
clones, each one representing 1000s of fragments (21).
Two other methods by which GFP could have been cloned include using an LIC system
and TOPO cloning. The first involves LIC Vectors Convenient pre-treated vectors
LIC Vectors are provided as ready-to-use, linearized vectors that have been treated with
T4 DNA Polymerase to produce non-complementary single stranded ends. Included with
each LIC Vector is a Positive Control Insert formed by annealing of two
oligonucleotides, that has single stranded ends compatible with the LIC Vector. Product
Description
pBAC"reg;-2cp is a baculovirus transfer plasmid designed for simplified cloning and
expression of target genes in insect cells. The plasmid is compatible with
BacVector"reg;-1000, -2000 or -3000 Triple Cut Virus DNA for low background
transfection and efficient utilization of the polh promoter. pBAC-2cp provides an ATG
start codon at the optimal position relative to native polyhedrin translation signals.
Cloning sites are provided for the creation of N-terminal fusions of an insert with
His*Tag® and/or S*Tag"reg; sequences. The vector provides optional expression of a Cterminal His*Tag fusion sequence by allowing read-through of inserts in the proper
reading frame (18).
The second is the TOPO cloning. Directional TOPO® Cloning (Figure 1) provides a
highly efficient, one-step cloning strategy to directionally clone a blunt-end PCR product
into an entry vector.
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Primer design is claimed to be easy and simple with this system. PCR product should be
amplified with a 4bp modification (CACC) on the 5' primer, the selected 3' gene specific
primer, and a proofreading enzyme. In addition, no ligase, post-PCR procedure, or
restriction enzymes are required. The pENTR/D-TOPO® and pENTR/SD/D-TOPO®
vectors are claimed to provide rapid, efficient cloning of your PCR products in the
correct orientation for downstream expression applications (9).
Another method includes Gateway Cloning Technology.
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The GATEWAY reactions are in vitro versions of the integration and excision reactions.
The goal is to move the gene of interest (or genes) from one vector backbone to another.
In the LR Reaction for making expression clones, the gene is cloned in an Entry Vector
that is transcriptionally silent, Km r , and is flanked by two recombination sites (attL1
and attL2). The gene must be moved to a Destination Vector backbone that contains all
the sequence information required for expression, and is Ap r . This plasmid also contains
two recombination sites (attR1 and attR2) that flank a gene for negative selection, ccdB.
Now combine the two plasmids and add LR CLONASE"reg; Enzyme Mix. Directionality
and specificity for recombination is conferred by att1 and att2 sites, so that attL1 only
reacts with attR1, and attL2 with attR2 (8).
The recombination reaction yields two constructs: the Expression Clone desired and a byproduct, labeled here as the Donor Vector. The expression plasmid is under two forms of
selection, antibiotic resistance and negative selection, supposedly ensuring a high level of
cloning efficiency. The "reverse" or BP Reaction can also be done by recombining DNAs
with attB and attP sites, respectively (8).
Excitation and Emission Spectra for
Living Colors"reg; Fluorescent Proteins (5).
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Keywords:
introduction polymerase chain reaction used amplify rare specific sequences into many
billions molecules when ends sequence known method amplifying rare sequences from
mixture numerous applications basic research human genetics testing forensics
application small samples such those found strand hair crime scene produce sufficient
copies carry forensic tests genomic digested into large fragments using restriction
enzyme then heat denatured into single strands synthetic oligonucleotides complementary
ends target segment interest added great excess denatured temperature lowered genomic
remains denatured because complementary strands concentration encounter each other
during period incubation specific oligonucleotides which very high concentration
hybridize with their complementary sequences genomic hybridized oligonucleotides then
serve primers chain synthesis which begins upon addition supply deoxynucleotides
temperature resistant polymerase such that from thermus aquaticus bacterium that lives
springs polymerase extend primers temperatures when synthesis complete whole mixture
heated further melt newly formed duplexes when temperature lowered again another
round synthesis takes place because excess primer still present repeated cycles cooling
melting heating quickly amplify sequence interest each round number copies sequence
between primer sites doubled therefore desired increases exponentially chain reaction
reaction elegant technique which fragments made replicate very rapidly illustrated
medical application early detection infection with virus that causes acquired
immunodeficiency syndrome aids sensitive detect very early stages disease before
symptoms appear only thousand blood cells patient infected with virus cloning
recombinant expression discovery types enzymes provided means common technique
cloning type called restriction enzymes from organism specific nucleotides generating
reproducible fragments other type called ligases insert restriction replicating molecules
producing recombinant recombinant molecules then introduced appropriate cells most
often bacterial cells descendants single such cell called clone carry same molecule once
clone bearing desired segment isolated unlimited quantities this prepared essence cell
chemistry isolate particular cellular component analyze chemical structure activity case
this feasible relatively short genomes small viruses genomes even simplest much large
directly analyze detail molecular level this obstacle obtaining pure samples large
genomes been overcome technology these methods virtually gene purified determined
functional regions explored altering planned ways reintroducing whole organisms
essence technology preparation numbers identical fragment interest linked through
standard phosphodiester bonds vector molecule replicate introduced host cell single
molecule composed vector plus inserted fragment introduced host inserted reproduced
along vector producing numbers include fragment originally linked types vectors most
commonly used escherichia coli plasmid vectors bacteriophage lambda vectors plasmid
replicate along their host while lambda lytic viruses killing packaging virions early
biochemists stanford university showed genetic traits could indeed transferred organism
another experiment microorganism recombined inserted another thus been edited exhibit
modification actual editing insertion process painstaking involves manipulating
incredibly tiny pieces incredibly tiny organisms process explained terms editing written
text scissors glue used paste methods rdna technology fairly simple take example
sentence gene insulin production humans paste coli bacterium inhabits human digestive
tract bacterial divide rapidly making billions copies themselves each bacterium carries
faithful replica gene insulin production coli inherited human insulin sentence transfer
embodying instruction production necessary appropriate paste splice plasmid special kind
takes circular form vehicle editing scissors class enzymes there well over hundred cutting
precise base these scissors singly various combinations segment specifies isolated glued
place using enzyme ligase result edited will able process instructions assemble amino
acids more importantly instructions passed along next generation known cloning
prokaryotic expression yeast other similar ideal purpose fact these contain necessary
normal eukaryotic transcription furthermore permit integration larger multicellular
organisms mammals possible result organism would transgenic transformed parent
produces transgenic offspring carrying integrated foreign parent expression allow
eukaryotic products detected prokaryotes bacterial usually plasmids strong promoters
ribosome binding sites transcription terminators eukaryotic proteins made inserting cdna
also antibodies screen clones cdna libraries order identify unknown genes proteins
eukaryotes since prokaryotes still able produce proteins glycosylation requirement some
have been established yeast similar ideal purpose fact contain necessary normal
transcription furthermore able integrated larger multicellular mammals possible result
would transgenic transformed parent produces offspring carrying integrated foreign cdna
libraries screened properties protein encoded cloned approach uses special lambda cloned
transcribed mrna turn translated encoded protein example phage have constructed
junction lies region transcribed translated high rate cloned position transcribed mrna
every infected type contains protein coding same reading frame infected will produce
fusion amino terminus encoded remainder molecular binds high affinity specificity
labeled probe identify clones expressing interacting affinity purification once clone
isolated separated cleavage enzyme form separated electrophoresis powerful method
separation according size electrophoresis also sued separate size estimate size nucleic
acid unknown length comparison migration known length highly charged near neutral
because phosphate group nucleotide contributes negative charge move toward positive
electrode during electrophoresis smaller move through matrix more readily than larger
different length separate matrix restricts random diffusion different separate bands whose
width equals well original mixture placed resolving power great stranded about
nucleotides long separated they differ only nucleotide composed electrophoretically using
polyacrylamide gels nucleotides agarose gels methods common visualizing bands
incubate solution containing fluorescent ethidium planar binds intercalating between base
pairs binding concentrated ethidium also intrinsic fluorescence illuminated ultraviolet
light regions containing fluoresces much more brightly than regions without affinity
purification specifically chromatography relies ability bind specifically columns packed
beads covalently attached ligand bind ligands substrates small bind widely form
technique chromatography attached ligand desired column will retain only ligand
attached beads remaining regardless their charge mass pass through column without
binding bound column eluted adding excess changing salt concentration obviously ability
particular depends selection appropriate ligands example certain want purify essentially
series histidines amino carboxyl terminus chelated activated nickel sticks eluted histidine
whose iminizole group serve elute fluorophore green fluorescent fluorescent
coelenterates pacific jellyfish aequoria victoria pansy renilla reniformis role transduce
blue chemiluminescence aequorin green light energy transfer become useful tool making
chimeric linked where functions tolerates terminal fusion broad variety expressed
bacteria yeast slime mold plants drosophila zebrafish mammalian noninvasive marker
living allows wide range applications where function lineage tracer reporter measure
interactions application involves visualization microtubule dynamics living plant green
higher plant show dynamic structural changes during cycle progression play significant
roles morphogenesis cortical preprophase band phragmoplast plant structures observed
interphase late phase prophase anaphase telophase respectively controls shape either
irreversibly reversibly orientating cellulose microfibril deposition wall involved
determining site division phragmoplast forms plate cytokinesis appearance disappearance
structures cycle have extensively studied immunofluorescence microscopy highly
synchronized tobacco studies were conducted revealed much about modes structural
organization dynamic changes cycle involves heart channel recent evidence suggests
biosynthesis rapidly modulated sympathetic interventions however data regarding
intracellular processing vivo lacking study sought establish model would allow study
subcellular localization model could eventually help better understanding trafficking vivo
potential role cardiac conduction terminus labeled construct compared transfected show
serve reservoir cardiac channels transport golgi apparatus among rate limiting steps
sarcolemmal channels materials stock reactions final volume buffer dntp microm target
pmol primer pmol pmol mgcl water above concentrations were prepared electrophoretic
purification extraction agarose etbr grams agaroseisopropanol waterelectrophoresis setup
edta tris acetic acid agarose melt microwave grams water about seconds until dissolved
melt contains edta tris acetic acid etbr always hind marker ladder loading tubes worry
contamination apply electric field visualize under light band purified sample different
buffers pull down water bath pieces melted liquid left tube comes right buffer
isopropanol precipitate create isopropanol naac product total pellet clear pellet dump
place clear pellet ethanol residual salt suck extra ethanol vacuum suction digest digest
buffer diluted diluted ulbam ecor eachwater mllb brotheppendorf tubes pipettes cutting
microgram ecor respectively ecor steam bath minutes digest hour bath kill eppendorf tube
ligase insert tube insert ligase transform ligation ligation control competent ligation
control placed styrofoam cooler rxns strain minsthen steam seconds heat shock room
minutes luria bertani broth minutes luria bertani consists tryptone pancreatic extract nacl
agar ampicillin platingdishesloopbunsen burnercontrol mixtures plates control plate
spread next plate spread last spread both centrifuge machinepipettestuberesuspension
solutionwatertrisbeefer reagentcuvette spin down resuspend smaller volume resuspension
solution contains rnase lysis solution naoh neutralization salt resuspend dave dilute ndei
xhoi clones multicore bamhi ecori stock tris test tubes resuspend pellets worth culture
cuvette original eppendorf beefer reagent bursts outer membrane lysozyme breaks
peptidoglycan layer down sonicate french press break rest open purificationresin
imidazoletriswatereppendorf tubespipettes incubate room break open step isolate target
subject hypotonic bursts soluble spin imidazole both imidazole bring resin spin invert
dump soluble contents resin take eppendorf pour rest resin aliquot supernatant imidazole
starting electrophoretic resolving mlstacking lower upper mlcis acrylamide mlaps temed
page based results discussion involved relying molecular techniques classical genetics
biochemistry microbiology initial stages straightforward involve isolation source
fragmentation relatively pieces combining replicates introducing screening hosts next
collected product hindi marker phage ladder contained base pairs purpose first purify
impurities negatively charged runs anode take both ends purifying there some things want
primers dntps confused presence dntps want supply substrate work dntps extend instead
doing phenol extraction avoid nonspecific product band after pairs buffers melted
precipitated important however expensive visualize products required loading glycerol
ficoll needed same they provide viscous medium does prevent diffusion however slows
rate diffusion load gels needed long needs elements creating include getting conducting
sticking design need origin replication determines copy number copy number exist
average within average copy ranges selective always lower left hand corner resistance
needs promoter terminator visualized under collected purified sample cutting bands need
retrieve placed containing piece melts liquid left comes right isopropanol precipitate spun
relative centrifugal force became clear dumped liquid ethanol residual extra vacuum
suction apparatus pneb polylinker bovine serum albumin must added reduce star activity
cleaves outside recognition conducted microgram added ecori heat inactivated conducted
hour kill piece alkaline phosphates takes phosphate dnma cannot ligate itself repair long
productse growth undigested undigested need inset than linear better circular ideal ration
here transformed colonies selected them grew them colonies every lane should positive
transformation competent styrofoam cooler competent reflect competency increase
competency exposing them cacl calcium phosphate reactions strain shocked seconds luria
bertani broth temporarily petri dish strains some strains include blue series strains hurts
there promoter produced since produced produced basic difference between former
promoter forms latter does indeed lactose repressor sits presence lactose repressor
inactivates instead lactose iptg analog membrane permeable conducting plating dishes
last were spun resuspended smaller volume colonies perhaps transform correctly class
possible positive reason class fact digested correctly carry following steps correctly
prepare design clean certain know properly assume clean killed certain whether worked
either transformation know certainty worked inoculation plating prep frozen stock lysing
wall naoh alkaline breaks wall neutralization contained well promotes hydrophobic
interactions binds beads crude prep precipitate recentrifugation done wash away nacl
promotes hydrophobic electrostatic interactions hydrogen bonds nacl affects star activity
stringency lowered performed dave electrophoretic done since excise just wanted look
usually among lowest most transcript alkaline lyses subject catalyzed hydrolysis received
fluorophores group received took worth culture spun resuspended cuvette bring original
around culture bursts outer membrane inner peptidoglyan lysozyme breaks peptidoglycan
layer usually sonication french press needed break rest open incubated room allowed
lyses soluble versus insoluble histaq free histidines chelate around nickel unlikely
histamines allowed make nickel chelates fool proof compete trying washed inverted
dumped contents took supernatant aliquoted took supernatant once again basically
spinning retrieving concentrations page sandwiched glycine mask charge properties
solely basis represent entire genome receive representing could system topo first
convenient treated provided ready linearized treated stranded included formed annealing
stranded compatible description pbac baculovirus transfer designed simplified genes
insect compatible bacvector triple virus background transfection efficient utilization polh
pbac provides start codon optimal position relative native polyhedrin translation signals
sites provided creation terminal fusions provides optional terminal fusion allowing read
inserts proper reading frame second topo directional topo figure provides highly efficient
step strategy directionally blunt entry design claimed easy simple system should
amplified modification cacc selected proofreading addition post procedure required pentr
pentr claimed provide rapid efficient your products correct orientation downstream
applications method includes gateway gateway reactions vitro versions integration
excision goal move genes backbone making entry transcriptionally silent flanked
recombination attl attl must moved destination backbone information required
recombination attr attr flank negative selection ccdb combine plasmids clonase
directionality specificity recombination conferred attl reacts attr yields constructs labeled
here donor under forms selection antibiotic resistance negative supposedly ensuring level
efficiency reverse done recombining dnas attb attp respectively excitation emission
spectra living colors
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