The staining solution is replaced the next day with destaining solution.Ģ1. The gel is transferred to the staining solution for overnight.Ģ0. The gel is removed with the help of spatula and the gel is marked by cutting the corner of the bottom side towards the last sample to have identity of samples.ġ9. It takes about 4-4.30 hours for complete run.ġ8. The run is stopped when the dye has reached approximately 0.5 cm from the bottom of the separating gel.ġ7. The run is performed at a constant voltage of 100 volts.ġ6. Protein molecular weight marker is used along with the samples.ġ5. The protein sample (5 µl) is loaded in the wells of the gel with a micropipette.ġ4. Electrode buffer is filled into the upper and lower chambers.ġ3. After the gel is completely set, the comb is removed carefully and the gel is transferred to the electrophoretic tank.ġ2. The gel is left for about one hour for complete polymerisation.ġ1. The stacking solution is poured between the glass plates and a comb with adequate number of teeth is inserted to make the wells.ġ0. The excess of water is removed from the top of the gel.ĩ. When the gel is fully set, a clear interface will be visible between the water layer and the gel.Ĩ. for polymerisation and the gel polymerisation can be easily known by checking the gel solution left in the beaker.ħ. Water is carefully over layered on the separating gel to have plain surface of the gel.Ħ. The separating gel is poured into the space between the plates leaving about 2.5 cm space from the top.ĥ. The second plate (without notch) is mounted over the first plate and both are clamped.Ĥ. The plate with notch is kept into the gasket and rubber spacer is fixed covering 3 sides (bottom, left and right sides).ģ. The glass plates are cleaned with water.Ģ. The gel casting is done in horizontal position of plates.ġ. The procedure described below is applicable to the Model AE 6210 slab gel cast and AE 620 dual slab chamber of ATTO Corporation, Japan. Protein sample is appropriately diluted with the sample buffer (30 µl: 30 µl) and heated in boiling water bath for 5 min. ![]() The supernatant containing protein is taken in eppendorf tubes and stored at 4☌/-20☌ till further use.ħ. The sample is centrifuged at 4☌ and 12,000 rpm for 35 min.Ħ. The sample is incubated in water-bath for 5 min. The sample is transferred to the eppendorf tubes (1.5 ml).Ĥ. 0.1 g seed is taken in precooled mortar and crushed with the help of pestle followed by addition of 1 ml extraction buffer.ģ. The seed coat is removed with the help of forceps.Ģ. Stacking gel (10 ml for 1 gel of 1 mm thickness)ġ. (To be added just before pouring the gel) Gel preparation separating gel – (20 ml for 1 gel of 1 mm thickness) ![]() This staining solution is stored in dark bottle. Solutions A and B are mixed and the volume is made up to 1 litre. For use, 1 part of this solution is mixed with 9 parts of distilled water. ![]() TEMED (N’N’N’N’ tetra methyl ethylene diamine)įinal volume is made up to 100 ml with distilled water and stored in dark bottle.Īfter adjusting the pH 8.8 with HCl/NaOH, the volume is made up to 100 ml.Īfter adjusting the pH 6.8 with HCl/NaOH, the volume is made up to 100 ml.įinal volume is made up to 100 ml with distilled water.Īfter adjusting the pH 8.6 with HCl/NaOH, the volume is made up to 1 litre. This solution is prepared afresh before use. Volume is made up to 100 ml and solution is stored in dark bottle.ġ0 g SDS is dissolved in distilled water and the volume is made up to 100 ml.ġ g APS is dissolved in 10 ml of distilled water. Volume is made up to 100 ml.Ģ g SDS is dissolved in 100 ml distilled water.ġ0 ml glycerol is mixed with 90 ml water and the final volume is made up to 100 ml.Ģ ml mercaptoethanol is mixed with distilled water and the volume is made up to 100 ml.ġ mM PMSF (phenyl methyl sulphonyl fluoride)ġ7.4 mg PMSF is dissolved in isopropanol. 12.14 g Tris HCl is dissolved in 80 ml distilled water and concentrated HCl is added until pH falls to 7.4.
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