8 Matching Annotations
  1. Jun 2019
  2. May 2019
    1. Plasmid DNA was isolated from10-100ml of bacterial culture grown overnight by using commercially available Miniprep or Midiprep kits (Invitrogen) as per manufacturers’protocol. Briefly, bacterial cells werepelleted by centrifugation at 6000 rpmfor10 min.The pelleted cells werere-suspended in 300μl-4ml of resuspensionsolution containing RNase H.About300μl-4ml of alkaline lysis solutionwas then added and mixed by gently inverting the tubesuntil clear and uniform lysatewas appeared. Subsequently, 350μl-5mlof neutralizing solutionwas added,the tubes were inverted repeatedly and gently to ensurehomogeneous mixing, followed by incubation for 5 min on ice. After centrifuging at 12,000 rpm for 15 min, supernatant was passed through commercially supplied filter columns by either gravity flow or high-speedcentrifugation. During this step, plasmid DNA binds to the column and was recovered by passing elution buffer through the column. The plasmid DNA present in the elution buffer was collected into a fresh tube and 70%(w/v)of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30 min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in desired amountof TE-buffer. Finally, the purity of plasmid wasobserved on 1% agarose gel
    2. Isolation of plasmid DNA
    1. The reaction mix was mixed well using a 200 μLpipette and 20μLeach was dispensed into eight 1.5 mL microfuge tubes. To this reaction mix 5μLof purified hexa histidine tagged IP6K1 enzyme (2-3 μg) was added. Tubes were placed in an acrylic box and incubated at 37°C overnight in a hybridization oven. The next morning, reactions were pooled into two 1.5 mL microfuge tubes containing 100 μLeach.100μLof 0.6 M perchloric acid was added to neutralise the reaction, the tubes were kept on ice for 1 min, and 33.5 μL of 1 M potassium carbonate with 5 mM EDTA was added and mixed by gentle tapping. CO2was liberated leaving a white precipitate, while tubes were kept open on ice for 1 h with gentle tapping at 15 min intervals.Tubes were centrifuged at 12000 x gfor 2-5 min and the supernatant from each tube was pooled into a new 1.5 mL microfuge tube
    2. to hold 1.5 mL microfuge tubes.Acrylic shield was used through out to block the β radiation
    3. Synthesis of 5[β-32P]IP7was conducted as described earlier (Azevedoetal., 2010). An acrylic box was placed at a 37°C in a hybridization oven before settingup the reaction
    4. Synthesis of radiolabelled 5-IP7