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9.
RESTRICTION DIGESTION OF VECTOR, INSERT ELUTION, LIGATION AND
TRANSFORMATION
Molecular cloning
is an important tool to understand and alter the structure, function and
regulation of individual genes and their products. Molecular cloning
involves the techniques for accomplishing DNA manipulations for a variety of
purposes.
Molecular cloning involves:
·
Isolation and digestion of Vector DNA
·
Elution of insert DNA
·
Ligation of vector and insert
·
Transformation into host cells
·
Confirmation of clones by plasmid isolation and restriction digestion
·
Expression of cloned genes in appropriate hosts
Restriction digestion of vector, insert elution & ligation:
Almost all vectors contain a closely arranged series of synthetic cloning
sites (polycloning site or multiple cloning site). These restriction sites
are unique and are not found elsewhere in the plasmid vector. Foreign genes
are cloned at these sites. Most plasmid vectors carry 2 or more selectable
markers, which help in identification of recombinant clones.
The vector selected for the cloning experiment here is Bluescript. This
vector has a multiple cloning site (MCS) and the lac gene. This gene can be
induced by isopropylthiogalactoside (IPTG) to express the first 146 aa of
the enzyme
β
galactosidase. The host cells express the carboxy terminal portion of
β galactosidase. Neither the host encoded nor
the plasmid encoded fragments of β galactosidase
are themselves active but, can associate to form an enzymatically active
protein. The active enzyme has the ability to hydrolyze the colourless
5-bromo 4-chloro 3 indolyl β D galactoside
(X-gal) and produce a blue colour. The MCS is present in the lac region.
Insertion of DNA in the MCS leads to loss of β
galactosidase activity and recombinant colonies appear white in colour. This
vector also has an ampicillin resistant gene for selection.
Digestion of Vector DNA:
Restriction enzyme digestions are performed by incubating double stranded
DNA molecules with an appropriate amount of restriction enzyme, in its
respective buffer as recommended by the supplier, and at the optimal
temperature for that specific enzyme. Typical digestion reactions include
required quantity of enzyme, required concentration of buffer, water and
known quantity of DNA. These reactions are incubated for 2-3 hr at 370C
to ensure complete digestion of DNA.
DNA VECTOR
DIGESTION
Reaction:
DNA 20ml(10mg)
Enzyme 1ml(10u)
Buffer (10X) 10ml
B S A
10ml
Sterile H2O
_ 59ml
Reaction
is set to 100
μl
Elution of
DNA using low melting Agarose:
Elution is
the process of recovery of desired restricted DNA fragment from the agarose
gels.
Materials
required:
1.
Low
melting agarose or sea plaque agarose.
2.
Tris
saturated phenol. (pH8).
3.
Chloroform: isoamyl alcohol (24:1).
4.
Absolute
ethanol.
5.
3M Sodium
acetate (pH 5.2).
Protocol:
1)
Gel
electrophorose the digested vector sample in a 0.8% Agarose gel.
2)
Run the
gel till the desired fragment separates finely by visualizing the gel under
UV light.
3)
Cut out a
block of Agarose with a scalpel blade, below the desired insert fragment.
4)
Now, fill
the gap with 0.8% Low melting Agarose (Sea plaque Agarose).
5)
Keep the
gel at 20 0C for the low melting agarose to solidify.
6)
After the
low melting agarose solidifies, resume the electrophoresis until the desired
insert fragment enters the low melting/sea plaque agarose block. See the gel
under UV light to check if the desired fragment has entered the low melting
agarose.
7)
Cut the
sea plaque agarose piece containing the desired fragment and take it into
an eppendorf.
8)
To this,
add 50 150 μl
of T10E0.1 (Tris 10 mM, EDTA 0.1 mM) depending on
the cut agarose piece.
9)
Keep the
eppendorf containing sea plaque agarose at 650C for 1015min to
melt it.
10)
To this,
add equal volume of Tris saturated phenol. Mix thoroughly and freeze the
mixture either in liquid N2 or by keeping it at -700C
for 15 20 minutes.
11)
Thaw the
mixture until it becomes liquid & centrifuge at 10,000 rpm for 5 min at 40C.
12)
Collect
the aqueous supernatent & mix thoroughly after adding equal volume of
chloroform: isoamyl alcohol (24:1). Extract by centrifuging at 10,000 rpm
for 5 min at 40C.
13)
Collect
the supernatent in an eppendorf and add 1/10th volume of 3M
Sodium acetate (pH 5.2) and 2.5 volumes of chilled absolute ethanol.
14)
Keep the
sample at -700C ultra low freezer for 30 min for precipitation.
15)
Centrifuge
the sample at 12,000 rpm for 12 min at 40C.
16)
Discard
the supernatent & wash the pellet with 70% ethanol.
17)
Dry the
DNA pellet at 650C for 5-10 min and dissolve it in 5-15
μl
of T10E0.1 at 650C.
18)
The DNA
concentration is estimated spectrophotometrically.
Preparation of Insert:
The target
gene is amplified by PCR Amplification with a DNA polymerase lacking 3-5
exonuclease activity (eg. Taq polymerase) yields products that contain a
single 3 terminal nucleotide overhang typically
dATP.
For
ligations involving PCR products various methods can be followed: The PCR
products can be conveniently cloned into a specially constructed vector
containing a single T overhang (eg. PGEM-T). The PCR product can be
partially filled in with dNTPs and Klenow fragment of E.coli, DNA polymerase
I under controlled conditions and blunt end ligation can be carried out.
Protocol
for Klenowing:
Reaction:
DNA 10ml
(5mg)
Klenow
buffer 4ml
Klenow
enzyme 1ml(5u)
dNTPs
1ml
Sterile water 24ml
Total
reaction 40ml
Procedure:
1.
DNA sample
klenowed for 45 min at 370C.
2.
Heat
inactivate at 700C for 12minutes.
3.
To the
sample add equal volume of phenol:chloroform:isoamyl alcohol(25:24:1).Mix
thoroughly and centifuge add 10,000rpm for 10 minutes
4.
To the
supernatant add 1/10th volume of sodium acetate and 3 volumes of absolute
alcohol.
5.
Keep at
-700c for 30 minutes
6.
Centrifuge
the sample at 12000 rpm for 12 min at 40c .
7.
Dry the
pellet at 650 c for 5-10 min and dissolve in minimum amont of
water.
Ligation
Ligation
of a segment of foreign DNA to a linearised plasmid vector involves the
formation of new bonds between phosphate residues located at the 5
termini of double stranded DNA and adjacent 3
hydroxy moieties.
The formation of phosphodiester bonds between adjacent 5-phosphate
and 3 hydroxy residues can be catalysed in
vitro by two different ligases E.coli DNA ligase and T4
DNA ligase. For most ligation reactions T4 DNA ligase is used. It
is a derivative of gene 30 of phage T4, purified from the
infected cells of E. coli. It utilizes ATP as the source of energy
and joins both blunt and cohesive ends of DNA efficiently. Bacterial DNA
ligase cannot join blunt ends.
Strategies for Ligation
Several strategies are available to ligate foreign DNA with the plasmid
vector. The choice among them depends on the nature of the termini of
foreign DNA fragment and the nature of restriction sites in the vector and
foreign DNA.
Fragments carrying non complementary protruding termini:
These are generated by digestion of vector DNA with two different
restriction enzymes depending on the sites generated at two ends of the
insert. The process is known as Directional cloning. This is by far
the most efficient method for cloning.
Fragments carrying identical (blunt end or protruding) termini:
Fragments carrying identical termini must be cloned in a linearised plasmid
vector bearing compatible ends. During ligation reaction, the concentration
of the two types of DNA must be carefully adjusted in order to optimize the
ligation products. Using higher concentrations of DNA and more ligase
facilitates blunt end ligations.
Dephosphorylation of plasmid DNA:
In simple ligation reaction involving single enzyme digestion, the problem
that is commonly encountered is the self-ligation of the vector DNA
fragments. The two strands of the plasmid vector carry 5
phosphate residues and the chance for self ligation increases as it is easy
for a terminus on one end of a DNA molecule. In order to avoid
recircularization of the plasmid, the 5
phosphates from both the termini of the linear DNA are removed with alkaline
phosphatase or calf alkaline phosphatase.
Vector:Insert ratio:
After the vector and insert DNA have been prepared for ligation, the
concentration of each is estimated by agarose gel electrophoresis along with
the molecular weight standards of known concentrations. In most cases either
a 1:1 or 1:3 molar ratio of vector: insert works well.
Materials required for Ligation:
Enzymes:
T4
DNA ligase 1ml(5u)
T4
polynucleotide kinase 1ml(5u)
Ligation
buffer: 300mM Tris HCl pH7.5 1ml
100mM MgCl2
100mM DTT
10mM ATP
vector
1m1(.5mg)
Insert 3ml(1.5mg)
Sterile
water 3ml
Total
reaction 10ml
Transformation:
Transformation of E. coli is an essential step in many cloning
experiments. A simple moderately efficient transformation procedure for
E.coli involves usage of calcium chloride. Mandel & Hoga (1970) found
that treatment with CaCl2 allowed E. coli cells to take up
DNA from bacteriophage. Later Cohen et al (1972) showed that CaCl2
treated E. coli are also effective to take up plasmid DNA. The
present protocol is a modification of Cohen et al (1972) method, wherein
competent bacteria prepared in batches yield 5 x 106 to 2 x 107
transformed colonies per microgram of supercoiled plasmid DNA. E. coli
cells & plasmid DNA interact productively in an environment of divalent
cations and low temperature (0 - 50C). A brief heat shock
stimulates the actual uptake of DNA.
Every Bacterial transformation includes positive controls to measure the
efficiency of transformation, and negative controls to eliminate the
possibility of contamination & to identify potential causes of failure.
Negative control is an aliquot of competent cells with no DNA. Hence with no
selectable antibiotic resistance colonies do not develop. Positive control
is an aliquot of competent cells with a known amount of a standard
preparation of circular superhelical plasmid DNA used to measure the
transformation efficiency.
Materials Required:
1.
Media
LB Broth/Litre
Bactotryptone 10 gm
Yeast
extract 5 gm
NaCl
10 gm
Adjust pH to 7.0 with NaOH and make upto 1000 ml with water and autoclave.
LB Agar LB Broth + 1.5% Agar
Solutions:
·
Ampicillin
solution: Prepare a stock of Ampicillin (50mg/ml) in sterile water.
·
0.1 M CaCl2:
Prepare 10 ml aliquots of 1 M CaCl2 and store at -200C.Dilute
from this whenever needed.
·
Make a
stock solution by dissolving 20mg X-gal in 1 ml of dimethy formamide .Wrap
in aluminium foil and store at D -200 C
·
0.6 gms of
IPTG is dissolved in 25 ml of water.
Protocol:
1)
Pick up a single colony from a freshly grown plate of E. coli Top 10
cells & transfer into a 50 ml of LB broth. Allow it to grow on a shaker
set at 200 rpm and 370C.
2)
Innoculate 100
μl
of overnight culture into 10 ml of LB media. Incubate at 370C
with vigorous shaking and allow the cells to grow to log phase.
3)
Transfer the culture into a centrifuge tube under asceptic conditions.
4)
Centrifuge at 4000 rpm for 10 min at 40C.
5)
Decant the
media and suspend the cells in 4 ml of 0.1M ice-cold CaCl2.
Incubate on ice for 10 min at 40C.
6)
Centrifuge at
4000 rpm for 10 min at 40C.
7)
Decant the
supernatant and repeat the suspension of cell pellet in 0.1M ice cold CaCl2
twice.
8)
Centrifuge at
4000 rpm for 10 min at 40C.
9)
Finally
suspend cell pellet in 400
μl
of 0.1M ice cold CaCl2 solution
10)
Aliquot 50
μl
into 1.5 ml eppendorf tubes. Use the required cells & store the rest at -700C
by adding Dimethyl sulfoxide (140 μl/4 ml of
suspended cells).
11)
Add 5
μl
of ligation mix to one tube and set aside two tubes as controls (one
positive and one negative). To the negative no DNA is added and to the
positive, add a known concentration of DNA.
12)
Incubate
the samples on ice for 30 mins at 40C.
13)
Keep the
tubes in a circulating water bath at 420C for 90 secs.
14)
Immediately transfer the tubes onto ice to chill for 1-2 mins.
15)
Add 750
μl
of LB Broth to each tube and incubate in a shaker at 370C for 45
min-1 hour
16)
Centrifuge
the cells at 4000 rpm for 10 min at 40C.
17)
Decant half the supernatent and plate the cells on selection plates (first
negative, followed by ligation mix and positive control).
18)
Incubate the plates in an inverted position for 12-16 hours at 370C.
Blue white Screening:
1)
To a LB agar plate containing ampicillin(50mg/ml), add 40
μl
of X-gal (20 mg/ml stock) and 100 μl of a solution
of 0.1M isopropyl this-B-D-galactoside (IPTG).
2)
Using a sterile glass spreader spread the solution over the plate. Incubate
the plates at 370C until all the fluid disappers.
3)
Incubate the plates with bacteria to be tested. This is done by streaking
with a bacterial loop or spreading upto 100
μl
of suspension of bacterial culture over the surface of agar medium.
4)
Allow the inoculum to be absorbed and then incubate the plates in an
inverted position for 12-16 hrs at 370C.
5)
Store the plate at 40C for several hours for the blue colour to
develop.
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