3. MATERIALS AND METHODSshodhganga.inflibnet.ac.in/bitstream/10603/6397/8/08_chapter 3.pdf · Fig....

Investigations on the Biotechnology of Microbial Alkaline Phosphatases from North Gujarat Soils

23 Ph.D Thesis Biotechnology Kadi Sarva Vishwavidyalaya, Gandhinagar

3. MATERIALS AND

METHODS



3. MATERIALS & METHODS

3.1 Collection and analysis of Soil Samples

3.1.1 Geographical area of sampling sites

Patan is a district from North Gujarat situated between the North latitude 23°23’ to

24°9’ and between East longitude 71°2’ to 72°29’. Its East-West distance is 146.72 kms and

North-South distance is 84.568 kms. It is bound in South-West by the Little Ran of Kutch.

3.1.2 Collection of soil samples

3.1.2.1 Sample collection sites

Soil samples were collected from saline lake soils apart from normal cultivated soils

from Patan district in August, 2009 from different lake sites and waterbodies of Patan district

viz. site A: inside the Lodra lake, site B: bank of Lodra lake, site C: inside the Bhilot lake, site

D: bank of Bhilot lake, site E: inside the Santalpur lake, site G: inside the water body at

Radhanpur, site H: inside the Sami water body and site I: bank of Sami water body. The field

soil sample was collected from the farm site F of Gujarat State Fertilizers Company (GSFC),

Bhilot, Patan district. The sample collection sites are depicted in Fig. 3.1 and 3.2.

3.1.2.2 Collection, transportation and storage of samples

The sediment soil samples from lakes and waterbodies were collected by sterilized

scoop in sterilized polyethylene pouches / bottles in triplicate from each site. Samples from

each site were pooled to make composite sample. All samples were transported to the

laboratory in intact physical conditions. The soil samples were stored at 4°C.

3.1.3 Analysis of soil samples

All soil samples were serially diluted and checked for microbial counts, pH, Electrical

conductivity (E.C.) and Phosphorous content in triplicate and average values are presented.



Microbial counts (colony forming units per g = CFU/g) were enumerated by standard plate

count (SPC) technique and pH, E.C. and Phosphorous content were got analyzed at Soil testing

laboratory, Indian Farmers Fertiliser Cooperative Limited (IFFCO), Kalol.

Bank of Lodra lake Bank of Santalpur lake

Sami waterbody Bhilot lake

Bank of Bhilot lake Radhanpur waterbody

Fig. 3.1: Sampling sites at Patan district.



Fig. 3.2: Map of Patan district showing sample collection sites (Adapted from

www.mapsofindia.com, 2010).

3.2 Isolation and primary characterization of the microbes

3.2.1 Isolation of microbes*

Thornton’s medium (Bajpai et al., 1964) and Martin’s Rose Bengal Chloramphenicol

agar medium (Atlas, 2006) were used for isolation of bacteria and fungi respectively. The

isolates were purified by repeated streak-plate method on Nutreint agar medium / Potato

Dextrose agar medium respectively (Atlas, 2006).

* Media compositions were detailed in Appendix I.

3.2.2 Characterization of the microbes

Morphological characters of the all isolates were noted. Gram’s staining and

lactophenol cotton blue staining (Coppuccino, 1998) were performed for bacterial and fungal

isolates respectively.

3.3 Screening and characterization of ALP producers

3.3.1 Screening of ALP producers



The pure isolates of bacteria were cultured on plates with MG-PDP medium and those

of fungi on Modified dermatophyte test agar medium to check the ALP production. The MG-

PDP medium as well as the Modified dermatophyte test agar medium with variable pH (7.5,

8.0, 8.5, 9.0) were supplemented with Methyl Green dye (MG) 50 mg/ml and Phenolphthalein

diphosphate tetra sodium salt (PDP) 1 g/l. The colonies producing ALP got stained with deep

green color, whereas the non ALP producing colonies remained colorless. The intensity of the

green color for each isolate was noted and the isolates were graded according to the green color

intensity. Bacterial and fungal isolates with intense green colored colonies from lake soils as

well as field soils were selected for further work.

3.3.2 Characterization of ALP producers

3.3.2.1 Morphological and biochemical characterization of ALP producers

Characterization of all the bacterial isolates was carried out on the basis of Gram’s

Staining, Anthony’s method of Capsule staining and Schaeffer-Fulton method of Spore staining

and motility testing by Hanging drop technique (Coppuccino, 1998). Gram’s reaction of

isolates was confirmed by Vancomycin sensitivity using 30 µg sensitivity discs M/s Tulip

Diagnostics (P) Ltd., Goa, India. Biochemical characterization of the best isolates was

conducted by using HiAssorted biochemical test kit and HiBacillusTM identification kit M/s

HiMedia Laboratories, Mumbai, India and Staph Identification kit and Listeria Identification

kit M/s Tulip Diagnostics (P) Ltd., Goa, India.

3.3.2.2 Enzyme spectrum of ALP producers

The 10 best ALP producing isolates were also checked for the production of other

enzymes viz. Amylase, Protease, Lipase, DNAse, Lecithinase, Gelatinase and Oxidase on

suitable solid media e.g. Starch agar, Casein agar, Tributyrin agar, DNase test agar, Egg yolk

agar, Gelatin agar and Nutrient agar respectively. The isolates exhibiting substrate clearance

zones indicated the production of these enzymes.

3.3.2.3 ALP production in liquid medium

Inoculum preparation was carried out in 25 ml Nutrient broth (pH 9.0) in 100 ml

Erlenmeyer flask, incubated at 37°C and 120 rpm for 6 h to achieve optical density in the range

of 0.8-1.2 at 600 nm. 2% v/v inoculum from this growth was transferred to 50 ml of PDP broth

in 250 ml Erlenmeyer flasks and incubated at 37°C, 120 rpm for 48 h.



3.3.2.4 Utilization of organophosphorus insecticide by ALP producers

Utilization of organophosphorus insecticide by the best 10 ALP producer bacteria was

studied by growing the isolates in the A broth. The degradation of Acephate was checked by

means of removal of phosphate from it by Ascorbic acid method at 880 nm (Watanabe and

Olsen, 1965) and ALP activity was determined by the p-NPP method described earlier.

3.3.2.5 Intracellular and extracellular crude ALP determination

This fermentation broth was centrifuged at 7000 g for 15 min at 4°C. The cell free

supernatant was used as a crude extracellular enzyme preparation and the cell debris treated

with lysozyme was used as a crude intracellular enzyme preparation. ALP activity was

measured spectrophotometrically by determining the release of p-nitrophenol (p-NP) from p-

nitrophenyl phosphate disodium salt (p-NPP) at 400 nm (Robert and Evan, 2003; Garen and

Levinthal, 1960; Zappa et al., 2001). 100 µl cell free supernatant was added to 1000 µl of p-

NPP solution (1.35 mM in 50 mM Tris-HCl buffer at pH 9.0) and the mixture was incubated at

37°C for 10 min. One unit of enzyme activity is the amount of the ALP catalyzing the

liberation of 1 µM of p-NP per min. The experiments were conducted in triplicate and average

values were represented.

3.4 Selection and identification of best ALP producer

3.4.1 Growth pattern

To determine the growth pattern, 5% v/v of inoculum from actively grown parent

culture (A600 = 0.8 - 1.2) was inoculated into sterile 100 ml N. broth in 250 ml Erlenmeyer flask

and incubated at 37°C and 120 rpm. O.D. at 600 nm was checked in UV-visible

spectrophotometer at a gap of 30 min until the culture reaches stationary phase.

3.4.2 Sensitivity to antibiotics

The sensitivity of 2 selected strains against various antibiotics was determined by disc

diffusion method using antibiotic sensitivity discs and Mueller Hinton Agar (MHA) M/s

HiMedia laboratory, Mumbai. 0.2 ml of 100 times diluted log phase culture of each isolate was

spread on the surface of MHA plates, plates were allowed to dry and antibiotic discs were

placed by the dispenser provided with the antibiotic kit. The plates were checked for



susceptibility after incubation at 37ºC for 24 h as per the chart provided with the antibiotic kits

by HiMedia.

3.4.3 Scanning electron microscopy (SEM) of best ALP producer FPB17

Three-dimensional SEM images for judging the surface structure of the bacterial cells

were studied by preparing a thin smear of overnight culture on a coverslip and dried by blowing

air for 10-15 min. The smear was fixed with 2 % Glutaraldehyde solution and kept for 10-40

min. Post fixation, the fixative was removed by tilting on tissue and washing cover slip with

sterile distilled water followed by air drying. The cover slip was stuck on SEM holder by using

carbon conducting tape and placed in sputter coating unit. The samples were then coated with a

thin layer of gold (~ 50 or 100 microns) and then analyzed by SEM which was performed at 30

kV accurate voltage on a SEM model number LEO s- 440i. Images of FPB17 were taken in

6000x and 9000x magnification.

3.4.4 Molecular characterization of FPB17

DNA was isolated from the pure culture of FPB17 isolate. Its quality was evaluated on

1.2% Agarose Gel (Sambrook et al., 1989). Fragment of 16S rRNA gene was amplified by

Polymerase Chain Reaction (PCR) from the above isolated DNA. The PCR amplicon was

purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR

amplicon was carried out with 8F and 1492R primers using BDT v3.1 Cycle sequencing kit on

ABI 3730xl Genetic Analyzer. Consensus sequence of 16S rRNA gene was generated from

forward and reverse sequence data using aligner software. The 16S rRNA gene sequence was

used to carry out BLAST with the help of NCBI Genbank database. Based on maximum

identity score first ten sequences were selected and aligned using multiple alignment software

program Clustal W. Distance matrix was generated using RDP database and the phylogenetic

tree was constructed using MEGA 4. The sequence was submitted to NCBI GenBank.

3.4.5 ALP production by FPB17

FPB17 strain, an isolate from soil sample of lake located in Bhilot village was

maintained on nutrient agar slants. Inoculum preparation and ALP production were also carried

out in nutrient broth with initial pH 9.0. Inoculum was developed by transferring single colony

from the grown culture in 25 ml nutrient broth in 100 ml Erlenmeyer flask and incubation at

37°C and 120 rpm for 6 h to achieve optical density in the range of 0.8-1.2 at 600 nm. 2% v/v



inoculum was transferred to 50 ml of nutrient broth in 250 ml Erlenmeyer flask and incubated

at 37°C, 120 rpm on an orbital shaker.

3.5 Optimization of ALP production by FPB17

3.5.1 Optimization of incubation parameters for production of ALP

The effect of incubation parameters viz. pH (5–13), temperature (4–55°C), fermentation

time (12–96 h), inoculums volume (2-10% v/v) and shaker speed (120-180 rpm) on the

production of ALP was studied. The growth was measured as optical density at 600 nm after 24

h of incubation. 1 ml of the fermented broth was centrifuged at 7000 g at 4°C for 15 min and

cell free supernatant was used for determination of ALP productivity at 24 h intervals. The

experiments were conducted in triplicate and average values were represented.

3.5.2 Optimization of nutritional conditions for production of ALP by FPB17 using

response surface methodology (RSM)

3.5.2.1 Salt profile

The concentration of NaCl in the range of 0-12% in nutrient broth was studied

arbitrarily first. The selection of other significant metal salts with NaCl for production of ALP

was done by using RSM, a statistics based design which was applied in two stages, first to

identify the significant salts for production of ALP by using Placket–Burman design criterion

and later the significant salts deduced from Placket–Burman design were optimized by using a

central composite design. The experimental design and statistical analysis of the data were done

by using Design Expert software package (version-8.0.6.1), Stat-Ease Inc.

Placket–Burman design

Each variable was tested at two levels namely a high level denoted by (+1) and a low

level denoted by (-1) as listed in Table 3.1. Eleven variables were screened by conducting

twelve experiments and the experimental design was given in Table 3.2. All experiments were

conducted in triplicate and the average value of ALP yield was used for statistical analysis. The

variables, which were significant at 5% level (P < 0.05) from the regression analysis were

considered to have greater impact on ALP production and were further optimized using central

composite design.



Table 3.1: Level of metal salts used for the production of ALP by FPB17 using Placket–

Burman design criterion.

Factor Name Units Minimum Maximum

A NaCl g% 0.10 0.50

B MgCl2 g% 0.01 0.05

C MnCl2 g% 0.01 0.05

D ZnCl2 g% 0.01 0.05

E CoCl2 g% 0.01 0.05

F CaCl2 g% 0.01 0.05

G NH4Cl g% 0.01 0.05

H KCl g% 0.01 0.05

J CuCl2 g% 0.01 0.05

K FeCl3 g% 0.01 0.05

L Pb(NO3)2 g% 0.01 0.05

Table 3.2: Placket–Burman design of effect of metal salts for the production of ALP by

FPB17.

Run

order

A:

NaCl

B:

MgCl2

C:

MnCl2

D:

ZnCl2

E:

CoCl2

F:

CaCl2

G:

NH4Cl

H:

KCl

J:

CuSO4

K:

FeCl3

L:

Pb(NO3)2

1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1

2 -1 +1 +1 +1 -1 -1 -1 +1 -1 +1 +1

3 -1 -1 +1 -1 +1 +1 -1 +1 +1 +1 -1

4 +1 -1 -1 -1 +1 -1 +1 +1 -1 +1 +1

5 +1 +1 +1 -1 -1 -1 +1 -1 +1 +1 -1

6 +1 -1 +1 +1 -1 +1 +1 +1 -1 -1 -1

7 -1 -1 -1 +1 -1 +1 +1 -1 +1 +1 +1

8 -1 +1 -1 +1 +1 -1 +1 +1 +1 -1 -1

9 +1 +1 -1 -1 -1 +1 -1 +1 +1 -1 +1

10 -1 +1 +1 -1 +1 +1 +1 -1 -1 -1 +1

11 +1 -1 +1 +1 +1 -1 -1 -1 +1 -1 +1

12 +1 +1 -1 +1 +1 +1 -1 -1 -1 +1 -1



Central composite design (CCD)

CCD was applied to determine the optimum concentration of three most significant

metal salts (CaCl2, MgCl2, and KCl) other than NaCl screened from Placket–Burman design

criterion. The design with six start points and six replicates at the central point, resulting in 20

experiments was generated by Design Expert, Version 8.0.6.1, Stat-Ease Inc., Minneapolis

(MN) statistical software. The levels of factors used for experimental design are given in Table

3.1 and the coded variables are calculated according to the equation 1:

X = (X − X )∆X i = 1, 2, 3, … … . . , j − −− equation1

The behavior of the system was explained by the following second order polynomial

equation 2:

Y = b + b X + b X X + b X + e − − − equation2

Where Y is the predicted response, Xi, Xj are input variables which influence the response

variable Y; b0 is the offset term; bi is the ith linear coefficient; bii is the ith quadratic coefficient

and bij is the ijth interaction coefficient. Analysis of variance (ANOVA), regression analysis

were done and contour plots were drawn by using Design Expert.

3.5.2.2 C and N source profile

The effect of incorporation of different N-sources viz. urea, NH4Cl, (NH4)2SO4,

(NH4)2NO3, peptone, tryptone, yeast extract, meat extract and their combinations on ALP

production was studied arbitrarily first which was followed by pairing of significant N-sources

with different C-sources viz. glucose, fructose, ribose, sucrose, maltose, mannitol, starch,

sodium acetate, cellulose, sorbitol in 1:1 and 2:1 C:N ratio. The selected C and N-source

combination was optimized by using CCD as explained under 3.5.2.1.

The effect of cheap N-sources like Corn steep liquor (CSL) and Soya flour and cheap

C-sources like molasses and whey was also checked on the production of ALP.

3.5.2.3 Phosphate substrate profile

Various phosphate substrates [Nicotinamide adenine dinucleotide (NAD), Acephate,

Tricalcium phosphate (Ca3(PO4)2), Potassium dihydrogen phosphate (KH2PO4), Glucose-6-

phosphate (G6P), Phenolphthalein diphosphate (PDP)] were incorporated at different



concentrations (0.5, 2.5 and 5.0 mM) in the fermentation medium to analyze their effect on

ALP production.

3.5.2.4 Effect of non aqueous systems on ALP production by FPB17

The effect of non aqueous systems on the production of ALP was studied by

incorporating 10% v/v concentration of different solvents viz. benzene, acetone, methanol, 2-

propanol, ethanol and hexane in production medium.

3.6 Purification and characterization of ALP from FPB17

3.6.1 Purification of ALP from FPB17

3.6.1.1 Crude enzyme preparation

The culture FPB17 was grown in the medium optimized under 3.5 for 48 h at 35°C at

140 rpm. Then the fermentation broth was centrifuged at 7000 g for 15 min at 4°C. The cell

free supernatant is used as a crude enzyme preparation.

3.6.1.2 Ammonium sulfate precipitation

The crude enzyme was precipitated by 60% w/v of Ammonium sulfate. The precipitates

were stored overnight at 4°C and recovered by centrifugation at 7000 g for 15 min. The

supernatant was again precipitated by 15% w/v of Ammonium sulfate. The precipitates were

then resuspended in 50 mM Tris-HCl buffer, pH 9.0.

3.6.1.3 Dialysis

The protein precipitates were filled in dialysis bag and kept overnight in 50 mM Tris-

HCl buffer, pH 9.0 at 4°C. Then dialysis bag was then dipped in sucrose solution for 1 h to

remove salt, metal ions and water and to concentrate only proteins.

3.6.1.4 Ion Exchange Chromatography

The concentrated protein solution was loaded on a DEAE cellulose column (300 mm x

18 mm) pre-equilibrated with 50 mM Tris-HCl buffer, pH 9.0. The elution was carried out with

0.1 - 0.5 M NaCl in the equilibration buffer at a flow rate 0.2 ml/min. The protein content of

fractions was determined by measuring O.D. at 280 nm. The protein containing fractions were

assayed for ALP activity by p-NPP method at 400 nm.



3.6.1.5 Affinity Chromatography

The maximum ALP activity containing eluate fraction was further purified by affinity

chromatography. The stationary phase was prepared by coupling diazonium salt of 4-(p-

aminophenylazo)-phenyl arsenic acid to Sephacryl-200 HR as described by Brenna et al., 1975.

The fraction was loaded on a Sephacryl-200 HR column (200 mm x 10 mm) pre-equilibrated

with 50 mM Tris-HCl buffer, pH 9.0. The enzyme was eluted with a linear gradient of 0.0 - 0.2

M KH2PO4 in the equilibration buffer at a flow rate 0.9 ml/min. The protein content of fractions

was determined by measuring O.D. at 280 nm. The protein containing fractions were assayed

for ALP activity by p-NPP method at 400 nm.

3.6.2 Characterization of ALP from FPB17

The experiments were conducted in triplicate and average values were represented.

3.6.2.1 Immobilization of ALP from FPB17 The alginate entrapment of crude enzyme was performed according the method of

Johnsen and Flink, 1986.

2 ml of ALP crude enzyme was mixed with 20 ml of 2.5% sterilized alginate solution

and incubated for 30 min. at room temperature. After incubation, 3 ml of 2.5% Glutaraldehyde

solution was mixed and kept for 45 min at room temperature. The solution was added drop

wise with the help of sterile pipette into 0.5 M calcium chloride solution resulting in formation

of round beads of immobilized enzyme. Beads were then transferred to the glass column. The

substrate 1.35 mM p-NPP solution was then added to the column at a rate 0.5 ml/min and

samples were collected from the bottom of the column, after every 10 min interval. ALP

activity was measured by p-NPP method under 3.3.2.5. Graphical representation in the form of

time v/s enzyme activity (free/immobilized) was drawn to compare the ALP activity of free

enzyme with immobilized enzyme.

3.6.2.2 Determination of Molecular weight (M.W.) of ALP from FPB17

M.W. of ALP protein from FPB17 was determined by SDS-PAGE electrophoresis,

performed according to Laemmli, 1970 on a vertical slab 12% w/v separating polyacrylamide

gels at a constant voltage of 50-80 V for 5 h. Marker Proteins (10-225 kDa) by Novagen were

used as standard protein molecular weight markers. The gel was stained with 0.2% silver

nitrate.



3.6.2.3 Optima of pH of ALP from FPB17

The effect of pH on enzyme activity was studied by incubating the ALP from FPB17

with p-NPP substrate prepared in different buffers in the pH range 6-13 as per 3.3.2.5. The

buffers used were Citrate-Phosphate buffer (pH 6-7), Tris-HCl buffer (pH 7-9), Carbonate-

Bicarbonate buffer (pH 9-11), Na2HPO4-NaOH buffer (pH 11-12) and KCl-NaOH buffer (pH

12-13).

3.6.2.4 Optima of temperature and thermostability of ALP from FPB17

The temperature optimum for ALP activity was determined in the range 0-60°C at pH

9.0 and the ALP enzyme stability at different temperatures were studied by incubating the

enzyme in 1.35 mM p-NPP in 50 mM Tris-HCl buffer, pH 9.0 at different temperatures for 10

min, followed by the estimation of ALP activity at room temperature as per 3.3.2.5.

3.6.2.5 Substrate specificity of ALP from FPB17

1.35 mM solutions of different phosphate containing substrates including phenyl

phosphate, β-glycerophosphate, 2-napthyl phosphate, Adenosine monophosphate, D-Glucose-

6-phosphate, D-Fructose-1,6-diphosphate, Nicotinamide adenine dinucleotide and PDP were

prepared in 50 mM Tris-HCl buffer at pH 9.0 and checked for ALP activity in comparison with

p-NPP. The relative activity of ALP was determined.

3.6.2.6 Enzyme kinetics of ALP from FPB17

The effect of p-NPP on ALP activity was studied by incubating enzyme with different

concentrations of p-NPP (0.054, 0.081, 0.108, 0.162, 0.270, 0.540 and 1.080 mM) in 50 mM

Tris-HCl buffer, pH 9.0 at 35°C for 10 min. The Km and Vmax were determined from

Lineweaver-Burk plots (Lineweaver and Burk, 1934).

3.6.2.7 Effect of inorganic phosphate ion on ALP from FPB17

The effect of inorganic phosphate was determined by estimation of the activity in

presence of 0.00, 0.10, 0.25 and 0.50 mM KH2PO4. The Kis were determined from Lineweaver-

Burk plots (Lineweaver and Burk, 1934).



3.6.2.8 Effect of metal ions and EDTA on ALP from FPB17

The different metal ions viz. Mg+2, Zn+2, Ca+2, K+, Mn+2, Co+2, Sodium fluoride,

Sodium arsenate and EDTA in 0.1 and 1 mM concentration were incorporated in 50 mM Tris-

HCl buffer, pH 9.0 and relative ALP activity was estimated as compared to control which lacks

any metal ions.

3.7 Applications of purified ALP

3.7.1 Dephosphorylation of λ phage DNA

During ligation in vitro, DNA ligase catalyzes the formation of a phosphodiester bond

between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the

other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be

minimized by removing the 5´-phosphate residues from both termini of the linear, double-

stranded λ phage DNA with ALP. Materials required (buffers, reagents, enzymes and nucleic

acid) and their composition are detailed in Appendix II.

3.7.1.1 Restriction digestion of DNA

1. A reaction mixture of 20 µl of λ phage DNA with 25 µl of 2X assay buffer and 3 µl of

EcoR I / M1µ was prepared and incubated at 37°C for 1 h.

2. The mixture was exposed to 80°C for 20 min in a pre-set waterbath. Phenol:Chloroform

was added to remove the denatured enzyme.

3. DNA was recovered by standard ethanol precipitation. The pellet was rinsed with 1 ml of

70% ethanol and centrifuged for 2 min. 70% ethanol was removed from the supernatant

by allowing to evaporate. DNA pellet was dissolved in 50 µl of Tris-EDTA buffer (pH

8.0).

3.7.1.2 DNA dephosphorylation

4. 1 µl of B. flexus ALP (Specific activity 101.5 U/mg) and 49 µl dephosphorylation buffer

for this ALP was added to the digested DNA samples, and incubated at 35°C for 30 min

and then denatured at 60°C for 10 min.

5. 5 µl of Calf intestinal ALP M/s HiMedia (Specific activity 20 U/mg) and 45 µl 10X

dephosphorylation buffer for this ALP were added to the digested DNA samples, and

incubated at 37°C for 30 min and then denatured at 80°C for 10 min.



6. After denaturation, the ALP enzymes were removed by Phenol:Chloroform treatment.



by allowing to evaporate. DNA pellet was dissolved in 50 µl of Tris-EDTA (pH 8.0).

3.7.1.3 Ligation

8. 1 µl of T4 DNA ligase and then 10 µl of 2X assay buffer were added to the

dephosphorylated DNA samples and incubated at 16°C for 2 h in a pre-set waterbath for

ligation and then denatured at 65°C for 10 min.

9. After denaturation, the ligase enzymes were removed by Phenol:Chloroform treatment.



by allowing to evaporate. DNA pellet was dissolved in 50 µl of Tris-EDTA (pH 8.0).

3.7.1.4 Agarose gel electrophoresis (Sambrook et al., 1989)

11. After incubation period 5 µl of gel loading buffer was added to each sample.

12. The samples were then loaded onto 1% Agarose gel and electrophoresed at 100 V for 2 h

and visualized under UV-Transilluminator for locating the dephosphorylated DNA of λ

phage.

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