DNA isolation

Detection of beta-lactam antibiotic resistancegenes

Antibiotic resistance amongVibriofrom shrimp fa

Relative diversity and distribution of Vibrioin the water and sediment of Cochin estuary

Detection of toxR gene

Carbon source utilisation tes

Oxidative-Fermentative test

Sample collection

Carbon source utilisation t

Oxidative-Fermentative test

Sample collection

Active site identification, metal detection and interaction of Dof domain structure

Cis-regulatory elements analysis

Motif analysis

Evolutionary relationships of sorghum, rice and Arabidopsis with respect to Dofgene family

Gene structure prediction

Phylogenetic relationships among Dofproteins of sorghum

Chromosomal locations

In silico prediction of Dof gene family of sorghum

Genome wide identification and in silico characterization of Dof gene family of sorghum

Cloning of Dof genes of sorghum using pBSK vector

PCR amplification of Dof gene

PCR amplification of Dofdomain

PCR amplification of Dof domain and Dof genes

Active site prediction and docking study

Reaction resuspension

Transformation of ligated product in chemically competent E. coli host cells (DH5αααα strain)

Ligation reactions

Cycling condition

Spectrophotometric quantification of genomic DNA

Seed collection of different crops

Estimation of mRNA cytokines in rLdADHT+BCG vaccinated hamsters as well as in control groups

cDNA synthesis and amplification

NO production

Vaccination schedule and assessment of parasitic burden

Solutions used for cytokine assay

Assessment of Cytokine levels- IFN--12/IL-10 in lymphocytes of cured/endemic patients

Preparation of soluble L. donovani promastigote antigen

Polyclonal antibody generation

Recombinant protein Expression

Sequencing of ADHT gene

Restriction Digestion of Plasmid DNA

Colony PCR

Preparation of master plate and isolation of plasmid DNA from transformed E. coli (Mini Prep)

Confirmation of positive clone(s)

Elution of amplified gene from the Gel

Evaluation of QSAR models

Inhibitors Dataset

Molecular Docking

Assay for reduced glutathione(GSH)

Acridine orange/ethidium bromide (AO –EB) staining

Superoxide radical scavenging activity

FTIR analysis

GC-MS analysis

Algorithm

EM Procedure

Empirical Bayesian Smoothing

Haemocyte morphogenesis

Spreading inhibitory behavior

Inhibition of haemocyte aggregation

Haemolymph protein profile

Total haemocyte count assay

Immunomodulatory

Helicoverpa armiger

Spodoptera litura

Gut enzyme profile

Insecticidal activity

. Statistical analysis

Inhibition of haemocytes spreading behavior

. Inhibition of haemocytes aggregation behavior

Haemolymph protein profiling

Total haemocyte count (THC)

Haemolymph collection

Immunomodulatory

Lactate dehydrogenase

Asparate (AAT) and Alanine aminotransferase (ALT)

Gut enzyme profile

Oral toxicity bioassay

Microinjection bioassay

VS preparation

Insect collection

INSECTICIDAL AND IMMUNOMODULATORY ACTIVITY AGAINST INSECT PEST

Quantification of protein of VS yielded in the prey deprivation

Venomous saliva utilization

Maxillary stylet

Morphometry of head and stylet

Lactic dehydrogenase

Cholesterol content

Characteristic features ofLabeo rohita

Animal model for the study-Labeo rohita

Residual nutrients

Organic carbon

Soil sampling

Biomass and harvest inde

Yield and its component traits:

Phenological traits and plant height

Regression analysis

Statistical analysis

Kernel hardness

Coleoptile length(cm)

Plant height (cm)

pH

Identification of the strains

Tested individually

Increased Chemokine and Toll like receptor on T cells after vaccination in HBV positive newborns.

Cord Blood immune profiles of HBV positive newborns at birth(Cord blood vs. peripheral blood)

CD107a expression (marker of cytotoxicity)

HBV vaccination of the newborn

1X TBST

Immunoprecipitation and western Blotting

Medium and Serum

GFP-β-catenin- a.a

Cell Viability and IC50determination byMTT assay

Bacterial Strain

Cell Lines

Bacterialstrain

Procedure

Estimation

Calculation

Extraction and determination of sugar

Standard curve of sugar

Reagents

Estimation of total sugar

Extraction and determination of protein

Extraction and determination of ascorbic acid

LC 50 value

Dilution Formula

FACS buffer

Statistics

Cell lines

Plasmid mini preparation by alkaline lysis

X-gal

Flow Cytometric analysis of variant erythrocytes

Flow Cytometric analysis of variant erythrocytes

Aortic ring assay

Procedure

Reagent

Superoxide anion scavenging activit

Procedure

Physical Characterizatio

Single Cell Test

Electrochemical Measuremen

Physical Characterization

Catalyst Preparation

Materials

ASSESSMENT OF SEAWATER ELECTROLYSIS ON PLATNIZED TIN AND MAGNESIUM ALLOY NANOPARTICLES FORNON-MEMBRANE POWER SYSTEMS

Physical Characterization

Dissolved oxygen and Biological oxygen demand

. Extraction and isolation of embelin from E. ribes

High Performance Thin Layer Chromatography (HPTLC) analysis

pH values

Estimation of soluble proteins

Per cent Overlap:

somatotype Categories:

Mean Somatotypes:

Age Changes in' Somatotypes

Range of Component Ratings:

Range of Component Ratings:

Balanced ectomorph:

Isolation of inclusion bodies from E. coli cells

The a-PMB chain was subjected to acid-acetone treatment to separate the heme from the a globin. Briefly, a solution of concentrated a-PMB chain (5 ml; 30 mg/ml) was added dropwise to I 00 ml of thoroughly chilled acid-acetone solution (0.5% v/v HCI in acetone) with constant shaking, and then incubated at -20°C for 30 min to allow complete precipitation of the globin. The precipitated globin was isolated by centrifugation at 7000 rpm (4°C) for 15 min and the supernatant containing soluble heme was discarded

Preparation of heme-free a chain

enginethatistheproblembut,rather,theusersofsearchengineswhoare.Itsuggeststhatwhatismostpopularissimplywhatrisestothetopofthesearchpile

'Sam!' he called. 'Pippin! Merry! Come along! Why don't you keep up?'10There was no answer. Fear took him, and he ran back. As he struggled on he called again, and kept on calling more and more frantically. He was weary, sweating and yet chilled. It was wholly dark.'Where are you?' he cried out miserably.There was no reply. He stood listening. He was suddenly aware that it was getting very cold, and that up here a wind was beginning to blow, an icy wind. A change was coming in the weather. The mist was flowing past him in shreds and tatters. His breath was smok­ing.11 He looked up and saw with surprise that faint stars were ap­pearing overhead amid the strands of hurrying cloud and fog. Oat of the east the biting wind was blowing.'Where are you?' he cried again, both angry and afraid.'Here!' said a voice, deep and cold, that seemed to come out of the ground. 'I am waiting for you!''No!' said Frodo; but he did not run away. His knees gave,12 and he fell on the ground. Nothing happened, and there was no sound. Trembling he looked up in time to see a tall dark figure like a shadow against the stars. It leaned over him. He thought there were two eyes, very cold though lit with a pale light that seemed to come from some remote distance. Then a grip stronger and colder than iron seized him. The icy touch froze his bones, and he remembered no more.When he came to himself again, for a moment he could recall nothing except a sense of dread. Then suddenly he knew that he was imprisoned, caught hopelessly; he was in a barrow. A Barrow-wight had taken him, and he was probably already under the dreadful spells of the Barrow-wights about which whispered tales spoke. Hedared not move, but lay as he found himself: flat on his back upon a cold stone with his hands on his breast.As he lay there, thinking and getting a hold on himself, he no­ticed all at once that the darkness was slowly giving way:13 a pale greenish light was growing round him. He turned, and there in the cold glow he saw lying beside him Sam, Pippin, and Merry.There was a loud rumbling sound, as of stones rolling and fal­ling, and suddenly light streamed in. A low door-like opening appeared at the end of the chamber beyond Frodo's feet; and there was Tom's head against the light of the sun rising red behind him.'Come, friend Frodo!' said Tom. 'Let us get out on to the clean grass! You must help me bear them.' Together they carried out Merry, Pippin and Sam. To Frodo's great joy the hobbits stirred, robbed their eyes, and then suddenly sprang up. They looked about in amazement. 'What in the name of wonder?14 began Merry. 'Where did you get to, Frodo?''I thought that I was lost', said Frodo; 'but I don't want to speak of it.' But Tom shook his head, saying: 'Be glad, my merry friends, and let the warm sunlight heat now heart and limb! Cast off these cold rags! Run naked on the grass!'

A stock solution of xylose (1 mg mL-1) was prepared in distilled water. A dilution series ranging from 100-1000 μg mL-1 was prepared from the stock solution. To 1 mL of solution, 1mL of DNSA was added and kept in a boiling water bath for 10 min and then 400 μL of sodium potassium tartrate solution was added and kept it for cooling. The absorbance was recorded in a spectrophotometer (Shimadzu, UV-VIS) at 540 nm

Preparation of standard curve of xylose

Transformation of calcium-competent cells was carried out by the procedure detailed below: •The competent bacterial cells were thawed briefly and 200 μL of cells was mixed rapidly with plasmid DNA (10-50 ng) in fresh, sterile microcentrifuge tubes and maintained on ice for 30 min. A negative control with competent cells only (no added DNA) was also included. •Cell membranes were disrupted by subjecting cells to heat-pulse (42 °C) for 90 sec. •After heat shock, cells were incubated on ice for 5 min. •Cells were then mixed with 1 mL LB medium and incubated with shaking at 37 °C for 1 h. •For blue/white screening 40 μL of X-gal solution (20 mg mL-1 in dimethylformamide) and 4 μL of the IPTG (200 mg mL-1) was spread on LB-ampicillin (LB-amp) plates with a sterile glass rod. The plate was allowed to dry for 1h at 37 °C prior to spreading of bacterial cells. •Bacterial cells (100-200 μL) were spread and the plate was incubated at 37 °C for overnight. •White colonies were picked from the plates and suspended into LB-amp broth and cultivated to OD600=0.5

Transformation procedure

PurifiedDNA fragments of size 2-8 kb were ligated to the treated vector using a 1:3::vector :insert ratio in a volume of 10 μL. The total amount of DNA was about 0.5 μg. Vector and insert DNA was heated to 45 °C for 10 min and the immediately chilled on ice for 5 min prior to addition of ligase and buffer. T4 DNA ligase (NEB, England) was added to a final concentration of 0.125 UμL-1 and reactions were incubated at 16 °C for overnight in a ligation chamber. Reaction mixture incubated under same condition without addition of the enzyme was used as control. A ligation reaction was also set up under condition with linear plasmid DNA containing the

Ligation of insert DNA with dephosphorylated vector

In order to minimize self ligation of vector during cloning experiments, the digested DNA was subsequently treated with calf intestinal phosphatase (CIP) [NEB, UK]. The reaction conditions and amount of CIP were optimized and varied from (0.06-1) unit/picomole DNA termini. The dephosphorylation reaction was carried out in 50 μL reaction as follows. Reaction mixture containing no restriction enzyme was treated as control. Reaction was incubated for 1 h at 37 °C and stopped by heat inactivation at 65 °C for 20 min. 2.5.5. Composition of restriction mixture (50 μL) Linearized Plasmid DNA X μL (1 μg) CIP 1 μL (0.06-1 U μL-1) Reaction buffer (10X) 5.0 μL Distilled water Y μL Total volume 50 μL Linearized and dephosphorylated plasmids from each reaction were purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany). 100 ng DNA from each reaction was then ligated in15 μL reaction volume containing 1.5 μL of 10X ligation buffer (NEB, England) and 0.2 μL of T4 DNA ligase to check the efficiency of self ligation after dephosphoryaltion. The ligation mixture was incubated at 16 °C for overnight and transformed into E. coli DH5αcompetent cells.

Dephosphorylation of the restricted plasmid

The vector isolated as above was digested with BamHI to generate the cohesive ends. The reaction was performed in 1.5 mL Eppendorf tubes as described below. Composition of restriction mixture (100 μL) Plasmid DNA X μL (20 μg) Bam HI 8 μL (10 U μL-1) NEB buffer 4 10.0 μL BSA (100X) 1 μL MQ water Y μL The reaction mixture was incubated at 37 °C for 3 h. The digestion was stopped by heat inactivation at 65 °C for 20 min. The digestion of plasmid was checked using 1.2 % (w/v) agarose gel electrophoresis for linearization of the plasmid. The digested plasmid was purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany).

Restriction digestion of plasmid DNA

Two hundred μL of alkaline-SDS solution was added to the above suspension, mixed by inverting the tubes up and down 3 times and incubated for 5 min at room temperature. ƒTo the above mixture, 250 μL of 3 M Na-acetate (pH 4.8) was added, mixed by inverting the tubes up and down 3 times, and centrifuged at 12,000 x g for 10 min. ƒThe supernatant was collected in another micro centrifuge tube (MCT), 200 μL of phenol:chloroform solution was added, inverted two times and centrifuged at 12, 000 x g for 8 min at room temperature. ƒThe aqueous phase was transferred to new tubes and 500 μL of chilled (-20 °C) ethanol (96 %) was added. ƒThe tubes were centrifuged at 13,000 x g for 25 min at 4 °C, supernatant discarded and pellet dried for 15 min at room temperature. ƒThe pellet was washed with 500 μL of chilled 70 % (v/v) ethanol and centrifuged at 13, 000 rpm for 4 min at 4 °C. ƒThe pellet was dried at room temperature and dissolved in 50 μL of 1X TE buffer (pH 8.0) containing RNase and stored at -20 °C till further use.

The cells of E. coli DH10B having p18GFP vector were cultivated for overnight at 37 °C in LB medium containing ampicillin (100 μg mL-1). ƒThe E. coli culture having p18 GFP vector (~1.5 mL) was taken in Eppendorf tubes and centrifuged at 10, 000 x g for 5 min. ƒThe pellet was homogenized by vortex mixing in 100 μL of homogenizing solution

Plasmid isolation from miniprep method

The metagenomic DNA extracted from above defined protocol was digested with Sau3A1 at conditions optimized to generate maximum fragment in the size range of 2-6 kb. Different concentration (0.05 to 1 unit) of enzyme was used to optimize the digestion of 1 μg of DNA. Reactions were carried out in a final volume of 30 μl each in an Eppendorf of 1.5 mL. Reaction mixture (1 μg DNA having 3 μL NEB buffer 3 and 0.3 μL of 10X BSA) were kept at 37 °C for 10 min and stopped by heat inactivation at 80 °C for 20 min. Different digested reactions were checked for the desired fragments using 0.8 % (w/v) agarose gel electrophoresis. After optimization of DNA fragments for the appropriate size, a large scale digestion was carried out and the fragments (2-8 kb) were purified from low melting agarose gel using gel extraction method according to the manufacturer’s protocol (Qiagen gel extraction kit, Germany)

Insert DNA preparation

CONSTRUCTION OF METAGENOMIC LIBRARY

An attempt was made to study the effect of storage of DNA extracts on DNA yield and purity. The DNA extracts were centrifuged and the supernatants were dispensed into 2.0 mL Eppendorf tubes and stored at -20 oC for a month. DNA precipitation and its quantification were carried out at a week intervals.

Effect of storage on soil/sediment DNA extracts

The isolated DNA was diluted (1:100) with MQ. The concentration (mg mL-1) of the DNA [N] was determined spectrophotometrically by recording absorbance at 260 nm (A260) as: A260 = ε 260[N]where ε 260 is the extinction coefficient of DNA (50 for ds DNA) [N] = concentration (mg mL-1) of DNA The concentration of ds DNA [N] was calculated as [DNA] (mg mL-1) = A260/ε 260 [DNA] (μg mL-1) = A260 × 50 × dilution factor Purity of DNA was checked by measuring absorbance at 260 and 280 nm and calculating the A260/A280 ratio (Sambrook et al., 1989). A DNA sample was considered pure when A260/A280 ranged between 1.8-1.9. An A260/A280 < 1.7 indicated contamination of the DNA preparation with protein or aromatic substances such as phenol, while an A260/A230 < 2.0 indicated possible contamination of high molecular weight polyphenolic compounds like humic substances.

Determination of DNA quantity and purity

as well as commercial methods (MN kit, Germany; Mo-Bio kit, CA, USA; Zymo soil DNA kit, CA, USA) according to the manufacturer’s protocols and compared in terms of DNA yield and purity.

The soil DNA from Pantnagar and Lonar soil samples were also extracted by various manual (Desai and Madamwar, 2007; Agarwal et al., 2001; Yamamoto et al., 1998

Alternatively metagenomic DNA was extracted from the alkaline soil samples by using different commercial kits (UltraClean™, PowerSoil™ [Mo Bio Laboratories Inc., Carlsbad, CA, USA], Nucleospin kit [Macherey-Nagal, Germany] and Zymo soil DNA isolation kit [CA, USA]). The DNA was finally suspended in 100 μL of sterile Milli Q water for further analysis.

Commercial kits

Comparison of yield and purity of crude DNA

Soil (1 gm) was suspended with 0.4 gm (w/w) polyactivated charcoal (Datta and Madamwar, 2006) and 20 μL proteinase K (10 mg mL-1) in 2 mL of modified extraction buffer [N,N,N,N cetyltrimethylammonium bromide (CTAB) 1% w/v, polyvinylpolypyrrolidone (PVPP) 2% w/v, 1.5 M NaCl, 100mM EDTA, 0.1 M TE buffer (pH 8.0), 0.1M sodium phosphate buffer (pH 8.0) and 100 μL RNaseA] [Zhou et al., 1996] in 20 mL centrifuge tubes to homogenize the sample and incubated at 37 °C for 15 min in an incubator shaker at 200 rpm. Subsequently, 200 μL of 10% SDS was added to the homogenate and kept at 60 °C for 2 h with intermittent shaking. DNA was precipitated by adding 0.5 V PEG 8000 (30 % in 1.6 M NaCl) and left at room temperature for an hour (Yeates et al., 1998). The precipitated DNA was collected by centrifugation at 8000 x g at 4 °C. The supernatant was discarded and pellet was dissolved in 1 mL of TE buffer (pH 8.0) and then100 μL of 5 M potassium acetate (pH 4.5) was added and incubated at 4 °C for 15 min. The supernatant was collected after centrifugation at 8000 x g and treated with equal volumes of phenol: chloroform (1:1) followed by chloroform: isoamylalcohol (24:1) at 8000 x g for 15 min

PROTOCOL FOR OPTIMIZATION OF HUMIC ACID-FREE DNA FROM ALKALINE SOILS

cycles at 94°C for 30 s, 45°C for 30 s, 68°C for 2 min and final extension at 72°C for 10 min (see table 3.1). PCR products were cloned in pGEM-T easy vector (Promega) and the sequence for the cloned PfCDPK4 gene was obtained by automated DNA sequencing

The PCR reaction was carried out usmg Hi-fi Platinum Taq polymerase (Invitrogen) and primers PfCDPK4_F and PfCDPK4_R (see list II) with the following cycling parameters: 94°C for 2 min initial denaturation followed by 3

To obtain PfCDPK4 gene sequence, BLAST search was done usmg either i TgCDPK1 or the published sequence of other CDPKs in the P. Jalciparum genome sequence. An ORF on chromosome 7 exhibited significant sequence homology with other PfCDPKs. Subsequently, PlasmoDB annotation appeared in the public domain and the gene sequence PF07 _0072 matched with the PfCDPK4 sequence! identified by us. For PCR amplification, primers were designed on the basis of' nucleotide sequence of PFb7 _0072. Total RNA from asynchronous P. Jalciparum, cultures was isolated using RNA easy Kit (Qiagen, Germany) and was used to' synthesize cDNA for reverse transcription (RT). Both complimentary and genomic DNA were used as template.

Molecular Cloning of PfCDPK4

For cryopreservation of P, Jalciparum cultures, mostly ring stage parasites at a high parasitemia were obtained. The parasites were pelleted by centrifugation at 200 g for 5 min with minimum de-acceleration. To the pellet, 1.5 volume of the freezing solution (list I) was added drop-by-drop, while shaking the vial gently; the ad4ition was completed in ~ 1 min. The medium was then transferred into a sterile cryovial, which was stored in ~he liquid nitrogen tank

Cryopreservation of Plasmodiumfalciparum cultures

sodium sulphate, filtered and concentrated. The product was reconstituted in methanol and desalted using LH-20 sephadex column. The identity of the aldehyde was confirmed by TOF-MS and tandem mass spectrometric analysis using ESI-MS (API QST AR Pulsar i MSIMS, Applied Biosystems). Purification of peptide was performed using RP-HPLC. The aldehyde could be resolved from other impurities (including traces of alcohol, valeryl-FTAAlaninol) on Cl8 RP-HPLC column (7.8 x 300 mm, 125A, Waters) using a gradient of 0-48% B in 20 mins, 48% B in 40 mins and 90%B in 50 mins (A: water with 0.1% TF A and B: acetonitrile with 0.1% TF A) using a flow rate of 2 mllmin. The elution profile was monitored at 220 nm and the identity of the aldehyde was confirmed by mass spectrometric analysis

The aldehyde valery I-L-Phe-L-Thr-L-Ala-L-Alaninal (valery!-FT AAlaninal) was synthesized by Fmoc-solid phase solid phase chemistry using the Weinreb AM resin (Novabiochem, 0.63 mM/g) and automated peptide synthesizer (Advanced Chemtech. USA). Fmoc protecting groups of amino acids were removed by 20% piperidine in double distilled dimethyl formamide (DMF). A fourfold excess of respective amino acids were preactivated using HoBt (2 equivalents) in DMF and the coupling was catalyzed by diisopropylcarbodiimide (DIPCDI, 2 equivalents). After synthesis resin was dried with dichloromethane/DCM (3 X) and MeOH (3 X). The Thr side chain protecting group (tertiary butyl) was removed by treatment with 60:40, TF A: DCM, twice. The filtrates were discarded and resin was washed with DCM (3 X) and MeOH (3 X). The dried resin was suspended in tetrahydrofuran (3 ml) in a glass reaction flask (25 ml) under nitrogen, swelled with gentle stirring for 1 h, and then cooled to 0 °C. Cleavage of the peptide aldehyde from the resin was performed by adding lithium aluminium hydride (Aldrich. 2 M equivalents dissolved in THF) drop wise for 30 min at 0°C with constant stirring. The reaction was quenched with careful addition of KHS04 (saturated solution) and stirred until the solution reached room temperature. The resin was then filtered off and washed with DCM (3 X) and MeOH (3 X). The filtrate was treated with sodium potassium tartrate (saturated solution) and organic layer was extracted. This organic layer was dried over

Clzemical synthesis and purification of aldehyde valeryl-FTA-Aianina