3 Matching Annotations
  1. May 2019
    1. For analysis of viral DNA by dot blot, I X I o5 cells were seeded in each we11 of the 9~ well plate and infected in duplicate with 50 J.Ll of the plaque pick for I h, followed b: addition of 50 J.Ll of CM to each well. Infected cells were incubated for 5 days, afte which the culture supernatant was saved and ce11s were processed for dot blot analysi~ Ce11s were lysed with 200 J.LI of 0.5 M NaOH. The alkali was neutralized by addition o 50 J.LI of 4 M ammonium acetate. The nylon membrane was wetted in warm water an( washed in dot blot solution (1 M ammonium acetate, 0.02 N NaOH) and the cell lysatt was blotted on to the membrane using a dot blot apparatus (Bio-Rad), dried, UV eros: linked and processed for prehybridization and hybridization. For the isolation of total genomic DNA, cells infected in a 35 mm culture dish wen harvested 72 h post infection (pi) and treated with 400 J.LI of DNA extraction buffer(]( mM Tris HCI, pH 8, 0.6% SDS, 10 mM EDTA)·and 50 J.Ll of 20 mg/ml proteinase K a 37°C for 12-I6 h. The DNA was extracted twice with phenol:chloroform:isoamy alcohol (25:24: 1) and once with chloroform. For each extraction, the suspension wa! mixed by inverting the eppendorf and separated by centrifugation at 2,000 rpm for 3 mir in a microfuge. DNA was precipitated with I ml of 95% ethanol at -20°C for 4 h anc pelleted at 4,500 rpm for 20 min. The pellet was washed with 70% ethanol, dried anc resuspended in 50 J.LI of TE. DNA was digested with Hind III, resolved on a 0.8% agarose gel and processed for Southern blotting. Positive clones were amplified b) infecting cells at a multiplicity of infection (MOl) of ~1 for 10 days and the amplifiec virus. was titrated using a plaque assay. Sf9 cells were infected at -I 0 MOl fo1 expression of the r-proteins.
    1. Meancorpuscularhaemoglobinconcentration(MCHC)istheaverageHbconcentrationperunitvolume(100)ofpackedredcells(W/V).Henceitisexpresseding/1whichisthesameaspercent(%).ItiscalculatedbythefollowingformulaHbMCHC=—......x100(g/dl)PCV
    1. Oligonucleotides and PCR products were end-labelled using phage T4-polynucleotidekinase (PNK, New England Biolabs or Fermentas or Sigma) with 32P-γ-ATP. The radiolabelling reaction mixture (20μl) contained 1X of buffer provided by the company, 10 units of T4-PNK and 40μCi of 32P-γ-ATP. The reaction mix was incubated for 1 hrat 37ºC and the reaction was heat-inactivated at 65oC for 20 minutes. The labelled oligonucleotides and DNA fragments were purifiedby the Qiagen nucleotide removal kit. Labelling efficiency was checkedeither by using Geiger-Muller (GM) counter orusing liquid scintillation counter.For scintillation counting, 1μl of radioactive sample wasadded to the 5ml scintillation cocktail, and radioactivity count was determined in the 32P channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). Liquid scintillation cocktail consists of 5g PPO (2,5-diphenyloxazol) and 0.3g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene, adjusted to a volume of 1L in toluene