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Molecular Characterization Of Sorghum using

RAPD and ISSR Marker



A project report submitted to the Department of biotechnology
Lovely Professional University, Phagwara(Punjab)
In partial fulfilment of the Requirement for the Award of the
Degree of Bachelor of technology in biotechnology

Submitted by
Shaila Gupta (11004748)
B.tech(hons.) biotech
Under the guidance of Dr. Pankaj Kumar
Associate Professor & Officer In-Charge
Department of Biochemistry & Physiology
Principal Coordinator, Centre of Excellence in Ag- Biotechnology
College of Biotechnology, Sardar Vallbh Bhai Patel
University of Agriculture & Technology, Meerut, UP-250 110

CONTENTS

POLYACRYLAMIDE GEL ELECTROPHORESIS
Chemicals
Protocol
Pouring SDS polyacrylamide gels
Preparation of samples and running the gel-
Staining and de-staining of SDS polyacrylamide gel-
PLASMID DNA ISOLATION
Materials required
Alkaline Lysis Solution I
Alkaline Lysis Solution II
Alkaline Lysis Solution III
Alkaline Lysis Solution IV
Protocol
PREPARARTION OF COMPETENT CELLS
Chemicals
Preparation of solution
Protocol
TRANSFORMATION
Ligation reaction
Protocol
Selection of recombinant clone
RESTRICTION ANALYSIS
Protocol
COLONY PCR OF RECOMBINANT CLONES
Protocol










TABLES
TABLE 1: 5% gel (stacking gel)
TABLE 2: 12% gel (resolving gel)
TABLE 3: LIGATION REACTION
TABLE 4: RESTRICTION DIGESTION REACTION
TABLE 5: COLONY PCR



POLYACRYLAMIDE GEL ELECTROPHORESIS-

Almost all analytical electrophoreses of proteins are carried out in polyacrylamide gels under
conditions that ensure dissociation of the proteins into their individual polypeptide subunits
that minimize aggregation.

CHEMICALS:

30% acrylamide mix, 1.5M Tris (pH 1.5), 1.0M Tris (pH 1.5), 10% SDS, 10% ammonium
per sulfate, TEMED, staining solution, destaining solution and sterile distilled water.

TABLE 1: 5% gel (stacking gel)
1 ml 2 ml 3 ml
H2O 0.68 1.4 2.1
30% acrylamide mix 0.17 0.33 0.5
1.0 M Tris (pH 6.8) 0.13 0.25 0.38
10% SDS 0.01 0.02 0.03
10% ammonium sulfate 0.01 0.02 0.03
TEMED 0.001 0.002 0.003

TABLE 2: 12% gel (resolving gel)
5 ml 10 ml 15 ml
H20 1.6 3.3 4.9
30% acrylamide mix 2.0 4.0 6.0
1.0 M Tris (pH 6.8) 1.3 2.5 3.8
10% SDS 0.05 0.1 0.15
10% ammonium sulfate 0.05 0.1 0.15
TEMED 0.002 0.004 0.006

PROTOCOL:

Pouring SDS polyacrylamide gels-
The glass plates were assembled according to the manufacturers instructions.
Appropriate volume of resolving gel was prepared and the mixture was swirled
rapidly without any delay. Polymerization started as soon as TEMED is added.
The resolving gel solution was poured into the gap between the glass plates, ;eaving
sufficient space for stacking gel.
After polymerization is complete (30 mins), stacking gel is prepared and is poured
directly onto the surface of polymerized resolving gel.
Combs are placed carefully into the stacking gel and are removed after polymerization
is complete (30 mins).
The glass plates are assembled carefully at the appropriate position into the PAGE
unit.

Preparation of samples and running the gel-
While the stacking gel is polymerized, samples are prepare din appropriate volume of
1X SDS gel loading buffer and heated to 100C for 3 min to denature the proteins.
15l of each of the sample is loaded in a predetermined order into the bottom of the
wells.
The electrophoresis apparatus is attached to an electric power supply (the positive
electrode should be connected to the bottom buffer reservoir).
Voltage of 300-500 V and 15-30mA of current is applied for 3-4 hours.

Staining and de-staining of SDS polyacrylamide gel-
Coomassie brilliant blue is an aminitriarylmethane dye that forms strong but not
covalent complexes with proteins, most probably by a combination of vander waals
forces and electrostatic interactions with NH3+ groups.
The gel is carefully separated from the glass plates and immersed in at least 5
volumes of the staining dye in as staining box with continue shaking at room
temperature for overnight.
After complete staining, gel is immersed in at least 5 volumes of de-staining solution
and incubated at room temperature with continue shaking for 2-4 hours.
After de-staining, the gel cam be seen on white light in gel documentation system.

















PLASMID DNA ISOLATION
Bacterial plasmids are self replicating, circular extra chromosomal, DNA molecules. The
most convenient method for preparing plasmid DNA is alkaline lysis method. In this
procedure, cells are lysed by SDS at high pH and then neutralized. The plasmid DNA
reanneals rapidly while most of the chromosomal DNA and bacterial proteins precipitate as a
protein- DNA-SDS complex.
MATERIALS REQUIRED:
i. Alkaline Lysis Solution I
1. 50 mM glucose
2. 25 mM Tris-Cl (pH 8.0)
3. 10 mM EDTA (pH 8.0)
Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15
minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4C.

ii. Alkaline Lysis Solution II
1. 0.2 N NaOH (freshly diluted from a 10 N stock)
2. 1% (w/v) SDS
Prepare Solution II fresh and use at room temperature.

iii. Alkaline Lysis Solution III
1. 5 M potassium acetate, 60.0 ml
2. Glacial acetic acid, 11.5 ml
3. H2O, 28.5 ml
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store
the solution at 4C and transfer it to an ice bucket just before use.

iv. Alkaline Lysis Solution IV
1. Isopropanol

PROCEDURE :
Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at 12,000g for 30 seconds
4C in a microfuge. Store the remainder of the culture at 4C.
Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
Resuspend the bacterial pellet (obtained in step 2 above) in 100 l of ice-cold
Solution I by vortexing and then add 200 l of freshly prepared Solution II and
mix by inverting the tube rapidly five times so that all the pellet come in contact
with Solution II (do not vortex).
After five minutes add 150 l of Solution III vortex gently in an inverted position for
10 seconds to disperse Solution III through the bacterial lysate.
After 5 minutes centrifuge at 12,000g for 5 minutes at 4C in a microfuge. Transfer
the supernatant to a fresh tube.
Add equal volume of phenol:chloroform (1:1).Mix the organic and aqueous Phases by
vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4C in
a microfuge .Transfer the aqueous upper layer to a fresh tube.
Precipitate the double-stranded DNA with 2 voluume of ethanol at room temperature.
Mix by vortexing. Allow the mixture to stand for 2 minutes at room temperature
Centrifuge at 12,000g for 5 minutes at 4C in a microfuge
Discard the supernatant. Rinse the pellet of double-stranded DNA with 1 ml of
70% ethanol at 4C. Remove the supernatant, and allow the pellet of nucleic acid to
dry in the air for 10 minutes.
Redissolve the nucleic acids in 30l of TE (pH 8.0) containing DNase-free pancreatic
RNase (20 ug/ml).


















PREPARATION OF COMPETENT CELLS
For efficient uptake of foreign DNA, the bacteria have to undergo some form of physical
and/or chemical treatment that enhances their ability to take up DNA. Cells that have
undergone this treatment are said to be COMPETENT. In order to make bacteria take in the
plasmid, they must first be made "competent" to take up DNA.
Mandel and Higa (1970) showed that bacteria treated with ice cold solution of CaCl2 and
then briefly heated to 42C could be transformed with bacteriophage DNA. Exposure to
calcium ions renders the cells able to take up DNA, or competent. Heat shock facilitates
efficient entry of the plasmid into the cells.

CHEMICALS REQUIRED:

1. LB medium broth
2. 0.1M CaCl
2

solution
3. 0.1M MgCl
2

4. DMSO

PREPARATION OF SOLUTION:
To make 50 ml of 1M MgCl
2,
4.76gm of MgCl
2
was mixed in 50ml of water.
To make 20ml of 1M CaCl
2,
2.21 gm of CaCl
2
was mixed in 20 ml of water.
Autoclave the solutions.
In order to make 100ml of 80mM MgCl
2
and 20mM CaCl
2,
8ml of 1M MgCl
2
+ 2ml
of 1M CaCl
2
+ 90 ml distilled water were mixed.
In order to make 0.1 CaCl
2,
1ml of 1M CaCl
2
was mixed with 9ml of distilled water.
200ml of LB media was prepared by mixing 4gms of LB media in 200ml of distilled
water.
PROCEDURE:
A loopfull of glycerol stock of E.Coli DH5 was inoculated into 5 ml LB broth
incubated at 37
0
C overnight.
Incubate at 37
0
C.
The bacterial cells were transferred to sterile ice cold oakridge tubes.
The culture was placed on ice bucket for 10mins to bring the culture at 0
0
C.
The cells were recovered by centrifugation at 6500 rpm for 5 mins at 4
0
C.
Media was decanted from cell pellets.
The tubes were stood inverted on a pad of filter paper for 1 min to allow last traces of
media to drain away.
The pellet was resuspended by swirling genetic vortexing in 30ml of ice cold (80mM
MgCl
2
-20mMCaCl
2
).
The cells were recovered by centrifugation at 6500rpm for 5 mins.
The media was decanted from cell pellet.
The tubes were stood inverted on a pad of filter paper for 1 min to allow last traces of
media to drain away.
The pellet was resuspended by swirling or gentle vortexing in 2ml of ice cold 0.1M
CaCl
2
.
70L DMSO was added to the final volume and mixed by inverting the tubes 2 times.
Another 70L DMSO was added on and mixed as done before.
200ml of the sample was poured into microfuge tube.
Store at -80
0
C until further use.




















TRANSFORMATION:
LIGATION REACTION:
TABLE 3: LIGATION REACTION
Reagents Volume (l)
2X ligation buffer 0.5
vector 0.5
Pcr product(poly A tail) 3.5
T4 DNA ligase 0.5
Final 5.0

PROTOCOL:
Transfer 200l of each suspension of competent cells to a chilled sterile eppendorf.
DNA was added to each tube. The content was mixed by swirling gently.
The tubes were stored on ice for 30 mins.
The tubes were transformed to a pre-heated circulating water bath at 42
0
C. The tubes
were stored in this for 80 sec.
Tubes were transferred rapidly to an ice box. The cells were allowed to chill for 2
mins.
800l of LB media was added to each tube.
Cultures incubated at 37
0
C in a water bath for 45 mins to allow the bacteria to
recover.
Keep the culture in shaker incubator at 250rpm for 15 mins (37
0
C).
Before spreading the culture, 40l of X gal and 7l of IPTG were spread on the agar
plates.
Transfer the transformed competent cells(400l) in the agar medium in petri plates.
The agar medium contained ampicillin(100/ml) Pellet of cells was taken by
centrifuging at 10000rpm for 2 mins.
Culture was spread using sterilized rod.
Plates were inverted and incubated at 37
0
C.
Additional plates namely ampicillin positive and ampicillin negative were used as controls.


SELECTION OF RECOMBINANT CLONE:
The plates show two types of colonies blue and white, the blue colonies represent the false
positive result. The white colonies show true positive result and are used for further analysis.
RESTRICTION ANALYSIS:
This method was employed only to screen the clones carrying the amplified gene. The
isolated plasmid was digested to release cloned fragment by using ECoRI as follows:
In a microcentrifuge tube following ingredients were added in order-

TABLE 4: RESTRICTION DIGESTION REACTION
Chemical Quantity(l)
Nuclease Free water 7
Plasmid 10
Buffer ECoRI (10X) 2
ECoRI enzyme (10U/l) 1

PROTOCOL:

The contents were gently mixed by pipetting and incubated at 37C for 8 hrs.
The RE digestion was studied by running 1.2% agarose gel together with ladder along
with uncut plasmids.
The digested product was visualized and documented.













COLONY PCR OF RECOMBINANT CLONES:

PROTOCOL:
Take 5l of distilled water in a PCR tube.
Pick the white colony by touching the micropipettes microtip on the colony.
Mix this tip into the 5l of distilled water.
Repeat the steps 10 times for picking 10 colonies.
Out of the 5 l solution, 2 l was used for PCR.
The remaining 3 l solution was mixed into 10ml LB broth for growth.

The composition of the reaction is as followed:
TABLE 5: COLONY PCR
Components Standard quantity(l) Experimental quantity(l)
milliQ water 10.05 90.5
PCR buffer 1.5 15
dNTPs 0.3 3
DNA 1 20
Primer L 1 10
R 1 10
Taq Polymerase 0.15 1.5

The conditions for running the pcr were same as mentioned previously.
The recombinant colonies that were cultured in LB broth were plated on LB agar
plates containing ampicillin (100g/ml), Xgal (15l) and IPTG(2.3 l)