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-ChargeDepartment of Biochemistry & Physiology

Principal Coordinator, Centre of Excellence in Ag- BiotechnologyCollege 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 2.130% acrylamide mix 0.17 0.33 0.51.0 M Tris (pH 6.8) 0.13 0.25 0.3810% SDS 0.01 0.02 0.0310% ammonium sulfate 0.01 0.02 0.03TEMED 0.001 0.002 0.003

TABLE 2: 12% gel (resolving gel)5 ml 10 ml 15 ml

H20 1.6 3.3 4.930% acrylamide mix 2.0 4.0 6.01.0 M Tris (pH 6.8) 1.3 2.5 3.810% SDS 0.05 0.1 0.1510% ammonium sulfate 0.05 0.1 0.15TEMED 0.002 0.004 0.006

PROTOCOL:

Pouring SDS polyacrylamide gels- The glass plates were assembled according to the manufacturer’s 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. 15µl 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 glucose2. 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 4°C.

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 ml2. Glacial acetic acid, 11.5 ml3. 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 4°C and transfer it to an ice bucket just before use.

iv. Alkaline Lysis Solution IV1. Isopropanol

PROCEDURE : Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at 12,000g for 30 seconds

4°C in a microfuge. Store the remainder of the culture at 4°C. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible. Resuspend the bacterial pellet (obtained in step 2 above) in 100 μl of ice-cold Solution I by vortexing and then add 200 μl of freshly prepared Solution II and

mix by inverting the tube rapidly five times so that all the pellet come in contactwith Solution II (do not vortex).

After five minutes add 150 μl 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 4°C in a microfuge. Transferthe 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 4°C 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 4°C in a microfuge Discard the supernatant. Rinse the pellet of double-stranded DNA with 1 ml of 70% ethanol at 4°C. Remove the supernatant, and allow the pellet of nucleic acid to

dry in the air for 10 minutes. Redissolve the nucleic acids in 30μl 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 λ 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 broth2. 0.1M CaCl2 solution3. 0.1M MgCl2

4. DMSO

PREPARATION OF SOLUTION:

To make 50 ml of 1M MgCl2, 4.76gm of MgCl2 was mixed in 50ml of water. To make 20ml of 1M CaCl2, 2.21 gm of CaCl2 was mixed in 20 ml of water. Autoclave the solutions. In order to make 100ml of 80mM MgCl2 and 20mM CaCl2, 8ml of 1M MgCl2 + 2ml of

1M CaCl2 + 90 ml distilled water were mixed. In order to make 0.1 CaCl2, 1ml of 1M CaCl2 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 DH5α was inoculated into 5 ml LB broth incubated at 370C overnight.

Incubate at 370C. 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 00C. The cells were recovered by centrifugation at 6500 rpm for 5 mins at 40C. 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

MgCl2-20mMCaCl2). 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 CaCl2.

70µL DMSO was added to the final volume and mixed by inverting the tubes 2 times. Another 70µL DMSO was added on and mixed as done before. 200ml of the sample was poured into microfuge tube. Store at -800C until further use.

TRANSFORMATION:

LIGATION REACTION:

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

PROTOCOL:

Transfer 200µl 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 420C. 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.

800µl of LB media was added to each tube.

Cultures incubated at 370C in a water bath for 45 mins to allow the bacteria to recover.

Keep the culture in shaker incubator at 250rpm for 15 mins (370C).

Before spreading the culture, 40µl of X gal and 7µl of IPTG were spread on the agar plates.

Transfer the transformed competent cells(400µl) in the agar medium in petri plates. The agar medium contained ampicillin(100µ/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 370C.

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 REACTIONChemical Quantity(µl)Nuclease Free water 7Plasmid 10Buffer ECoRI (10X) 2ECoRI enzyme (10U/μl) 1

PROTOCOL:

The contents were gently mixed by pipetting and incubated at 37˚C 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 5µl of distilled water in a PCR tube. Pick the white colony by touching the micropipette’s microtip on the colony. Mix this tip into the 5µl of distilled water. Repeat the steps 10 times for picking 10 colonies. Out of the 5 µl solution, 2 µl was used for PCR. The remaining 3 µl solution was mixed into 10ml LB broth for growth.

The composition of the reaction is as followed:TABLE 5: COLONY PCR

Components Standard quantity(µl) Experimental quantity(µl)milliQ water 10.05 90.5PCR buffer 1.5 15dNTPs 0.3 3DNA 1 20Primer L 1 10 R 1 10Taq 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 (100µg/ml), Xgal (15µl) and IPTG(2.3 µl)