Team:Tec-Monterrey/projectprotocols

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1.Measure 60 ml of TBE (.5 X) with the probe.
2.Take a beaker and place 60 ml of TBE.
3.Place the filter paper on the scale.
4.Weigh 600mg of agarose gel on the scale.
5.Mix 600mg of agarose gel in TBE and heat it in the microwave for time intervals of 10 seconds.
6.Place the electrophoresis comb in the tray electrophoresis.
7.Pour the solution into the electrophoresis tray, making sure the level does not reach beyond the teeth of the comb. Wait until it solidifies.
8.Remove the electrophoresis comb and place the tray electrophoresis in the electrophoresis chamber.
9.Pour TBE by the sides of the electrophoresis chamber until the level of TBE exceeds the gel.
10.Mix the DNA samples, molecular weight ladder and DNA supercoil with 12μl of loading buffer and SYBR Green 4μl.
11.Load the wells with the samples made in the previous step.
12.Connect the electrophoresis chamber to the power supply at a voltage between 60V and 70V and run it for 1.5 hours.
13.Use transilluminator to see results.

1.Perform the necessary calculations using the calculator developed by the Tec-Monterrey 2010 team. (click here)
2.Add the required amounts of buffer, plasmid DNA, nuclease free water and BSA (keep reagents in ice except the sample DNA).
3.Pour the required amount of enzyme at the end.
4.Incubate for 1 hour at 37 ° C.

Soluble and insoluble protein extraction from bacterial cell culture & Preparation of samples for SDS-PAGE

6 ml of transformed cell culture is harvested by centrifugation at 10 – 12,000 x g for 1 min.
The supernatant is removed and the pellet is dried.
For each 1 g of cell pellet, 20 ml of xTractor Buffer, 40 µl of DNAse 200 µl of 100X lysozyme solution is added.
The suspension is incubated during 10 min at room temperature.
The crude lysate is centrifugated at 10 – 12000 x g for 20 min and the supernatant is called as soluble fraction and the pellet as insouble fraction.
The insoluble fraction is sonicated with water during about 5 sec.
50 µl of protein fraction is mixed with 50 µl of 2x sample buffer with 2-ME and heat 10 min at 95 °C.