· polyacrylamide gel must by acrylamide monomer, polymerization starting material, catalyst, and the right of salt and buffer mixture together.
· acrylamide and BIS (N, N ‘- methylene double acrylamide) is the monomer form gel matrix.
· ammonium persulfate start adhesive polymerization process. The formulation of the glue requires 10% ammonium persulfate solution prepared in water. Most of the information indicated a need for active use. However, the 10% solution can be placed at 4℃ for several weeks without significant loss of activity. Make up to 10 ml and discard when the glue fails to aggregate.
Tip: The percentage of acrylamide in sequencing glue and protein glue is not the same. If using premade acrylamide: BIS solution, make sure to get the right bottle.
· TEMED (N, N, N ‘, N ‘- tetramethyl ethylenediamine) is a catalyst, in a brown bottle, placed in the refrigerator. Add just before pouring glue.
· polyacrylamide electrophoresis used glass in electrophoresis should be washed before and after each time. After electrophoresis, wash with a soft brush and cloth in hot soapy water, rinse with distilled water and stand to dry.
· moisture and dust can cause with hollow polymer. Before electrophoresis, clean the glass plate with glass cleaner and wipe it with a soft brush. Wash with distilled water and dry thoroughly with a paper wipe. Rinsing with 70% ethanol before wiping with paper helps to clean and speed up drying. Add samples of acrylamide successively: BIS, water, buffer solution, ammonium persulfate, TEMED. Shake well and pour immediately.
It is not necessary to degass polyacrylamide before polymerization. (Acrylamide used to be placed in a vacuum to remove bubbles because oxygen inhibits polymerization.)
Horizontal glue point sample tips.
· put a piece of black paper in the bottom box, black background to make some sample hole to see more clearly.
· glue tank filled buffer, just over the colloid.
· if the edge has a light, turn on the lights, let light shines colloid. Draw the sample into the pipette.
· using automatic pipetting device.
· in 10-200 mu 1 liquid moving point can be used in most of the points on the sample. For very small sample holes (less than 10μ1), the long pipette head used for sequencing glue is more convenient.
· pipetting just immersed in the sample, inhaling slowly moving fluid. The sample may appear sticky with glycerin, and rapid pumping may draw air bubbles into the pipette head.
· samples after inhaling pipetting head, will move fluid head gently on the edge of the pipe or wiping paper suck liquid head outside of the droplets. Be careful not to suck the sample.
Place the sample into the sample hole
· pipetting device to keep a little pressure, make the samples slightly move overflow liquid head.
· the pipetting head inserted into the buffer, slightly higher than the spot holes, maintain positive pressure. The tip of the pipette can be inserted into a small hole.
· slowly and steadily the samples out. The pipette tip is placed above the point sample hole, and the sample will sink into the hole. Let the sample sink to fill the sample hole instead of pushing in.
· once the last drop of sample with liquid head, liquid will move to the second leg, slowly raise the liquid move, move out of buffer
How to do vertical glue sampling?
· vertical glue point sample holes formed between two pieces of glass. In very thin glue, the pipette head cannot even be inserted between two glass plates. Watch the glycerin! Put the pipette head over the sample hole and the sample will sink into the hole.
· point sample before, be sure to put the vertical point of polypropylene acyl gel sample hole is rinsed clean. Wash away the unpolymerized acrylamide and the water that may appear at the bottom of the sample hole. The water can make the sample hole significantly smaller. Use a 25ml or 50ml syringe and an 18-gauge needle. Pour in the electrophoresis buffer and carefully flush the sample hole.
· it can be difficult to see the sample hole, but at the same, the rest is easy. If there are excess holes, the sample buffer can be tested with bromophenol blue.
Post time: Mar-31-2023