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Wetlab Protocol

1. SDS PAGE
   

   SDS-PAGE analysis will running using 8-16 % Tris-glycine acrylamide gradient gels (Novex, San Diego, CA). The culture will prepared by mixing 25 µl of 2x loading dye with 25 µl of each cell suspension. Than the sample enter to the gel and also protein ladder. The solution will be heated at 100oC for 5 min. To each lane of gel, 10 µl of dye-sample mixture will be loaded and the electrophoresis perfomed in SDS-Glycine buffer at 130V constant until dye reached the bottom of the gel (Sambrook et al., 2003).



2. Ethylene Glycol Assay using Chromatic Acid
   1. Ethylene Glycol Standard Solution Procedure
      1. Ethylene Glycol concentration ≈ 0.5 M (31035 ppm) -> Dissolve 279 µL ethylene glycol in 10 mL deion water.
      2. Ethylene Glycol concentration ≈ 0.05 M (3103.5 ppm) -> Dissolve 300 µL ethylene glycol 0.5 M, then dilute until 3000 µL.
      3. Ethylene Glycol concentration ≈ 0.005 M (310.35 ppm)  Dissolve 300 µL ethylene glycol 0.05 M, then dilute until 3000 µL.
      4. Ethylene Glycol concentration ≈ 0.0005 M (31.035 ppm)  Dissolve 300 µL ethylene glycol 0.005 M, then dilute until 3000 µL.
      5. Ethylene Glycol concentration ≈ 0.00005 M (3.1035 ppm)  Dissolve 300 µL ethylene glycol 0.0005 M, then dilute until 3000 µL.
   2. Chromic Acid Standard Solution Procedure
      1. Chromic acid ≈ 0.6 M (177000 ppm)  Dissolve 1.77 gr K2CrO7 (Mr 294.18) with sulphate acid until the volume 10 mL.
      2. Do dilution of chromic acid same as ethylene glycol standard solution procedure.
   3. In your country, what are the regulations that govern biosafety in research laboratories? Please give a link to these regulations, or briefly describe them if you cannot give a link.
      Yes, our country does. It can be accessed in http://indonesiabch.or.id/tentang-bkkhi/