H. Isolation DNA from plasmid by gel electrophoresis
- Calculate the average concentrations
average concentrations = 90
How long microliters of samples
(3).(90)=270/1000=.27 1000 -- Is not a good choice because it is less than a thousand.
(10)(90)=900 /1000=.9 1000 -- Is not a good choice because it is less than a thousand.
(15)(90)=1350/1000=1.35 1000- -- A good choice because of it's biggest one thousand.
So ..
- 15 μL of sample.
- 5 μL of ECOR buffer.
- Enzyme ECOR 20000 n/ μL
Required/20000= result(1000)
1/20000=.00005 (1000)= .05 μL
- D.h2o 29.92 μL
- Total 50 μL
- Incubation by thermostat 37 °Cat 60min.
- Preparation of Gel Agqros 2%
100 ml of t- buffer
2 gram Agaros
- Heated in the microwave for two minutes, and the cover is not closed tightly.
- pPlace gel agarose in a hot water bath at 50 °C to the time of used.
- Pour the agarose into a gel tray with the well comb in place.
- Note: Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.
- Place newly poured gel at 4°C for 10-15 minutes OR let sit at room temperature for 20-30 minutes, until it has completely solidified.
- Loading Samples and Running an Agarose Gel:
- Fill gel box with 1xTAE (or TBE) until the gel is covered.
- Carefully load your samples and ladder into the additional wells of the gel.
- Using the DNA ladder in the first lane as a guide.
- Once you have run your gel, move it to an open UV box.
- Turn off UV.
- File – Gel Dou – manual or Automatic – freeze – print video.