Dna molecular weight marker

Conventional RT-PCR was employed for detection and serotyping of FMD in 51 field samples, using QIAamp viral RNA mini kit (Cat# 52904, QIAGEN, Crawley, UK). The extracted RNA was first tested by universal FMDV primers, targeting 5 UTR region according to Reid et al. [21 (link)]. The RT-PCR was performed using Verso^™ one-step RT-PCR kit according to the manufacturer instructions. Consequently, positive samples by universal RT-PCR were tested by serotype-specific primers for O and A according to Reid et al. [21 (link)] and SAT2 according to Ahmed et al. [14 (link)]. The PCR products were analyzed on 1.5% agarose gel containing 0.5 ug/ml of ethidium bromide (Sigma, USA). The DNA band was visualized by UV illumination, and the size was determined against DNA molecular weight marker (Fermentas, Germany).

DH5α and BL21(DE3)pLysS strains of E. coli are maintained as laboratory stocks. Plasmid pYUB28b and strain Mycobacterium smegmatis mc² 4517 were a kind gift from Dr. Ghader Bashiri, New Zealand [18 ]. Oligonucleotide primers were synthesized on order, in desalted form, by Sigma–Aldrich Chemicals Pvt. Ltd., India.
Restriction endonucleases, T4 DNA ligase, DNA molecular weight marker, dNTPs, Pfu DNA polymerase, polynucleotide kinase, DNA ligase and Taq DNA polymerase were procured from MBI Fermentas, Germany. Protein molecular weight markers were procured from Bangalore Genei or Sigma Aldrich Chemicals Pvt. Ltd., India. Agarose, calcium chloride, PMSF, DTT, acrylamide, N,N^′-methylene-acrylamide, Coomassie brilliant blue-G and -R, APS, TEMED were procured from Sigma-Aldrich Chemicals Pvt. Ltd., India. Thrombin was purchased from Novagen, USA. Streptococcus thermophilus UDP-Gal 4-epimerase was purchased from Calbiochem, USA. Antibiotics and IPTG were from MP Biomedicals, India or Himedia, India. Culture media were from Himedia, India. Qiaexpress Ni-NTA spin kits were procured from Qiagen, USA. All other chemicals were of analytical reagent grade procured from Sisco Research Laboratories, India or Himedia, India. Protease inhibitor cocktail tablets were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India.

l-Asparaginase biosynthesis gene (ansA) of S. griseus NIOT-VKMA29 was PCR amplified by using gene specific primers designed using Primer3 programme available at http://frodo.wi.mit.edu/primer3 (Table 2). PCR was performed in 50 μL of reaction mixture, which contained 50 ng of genomic DNA, 0.5 μM of each primer, 200 μM each of dNTP (MBI Fermentas), 1.25 U of Pfu DNA polymerase (MBI Fermentas), 1× Pfu buffer; 2.5 mM of MgSO₄ and for the rest autoclaved Milli Q water. Amplification was performed in a Master cycler (Eppendorf, Germany) with the following conditions: initial denaturation at 94 °C for 3 min, followed by 30 repeated cycles of 94 °C for 30 s, 52 °C for 1 min, 72 °C for 2 min and final extension at 72 °C for 10 min. PCR amplicons were analyzed on 1.5% agarose gel along with DNA molecular weight marker (MBI Fermentas) and documented in gel documentation system (UVP BioSpectrum Imaging system, USA).