For the last two classes we have being completing practical work using the Lambda Protocol. A quick summary of the protocol is that the lambda DNA was cut with different restriction enzymes. Then it was mixed with heavy dye solution. The negatively charged DNA fragments move towards the positive electrode. The gel becomes gained and reveals the DNA fragments. This, however, is all done in steps.
1) Add 100 micro litres of distilled water to a tube of the lambda DNA and mix around using the micro syringe.
2) Cap the tube and flick the bottom of it to mix the contents more.
3) Add 20 micro litres of the lambda DNA to an enzyme filled tube and mix.
4) Repeat this for the other 3 enzyme tubes.
5) Close each tube tightly and place in a holder in an incubator or water bath at 37 degrees celsius for between 30-45 minutes.
6) Melt some agarose gel in hot water. Once completed store in a water bath of between 55-60 degrees celsius until needed.
7) Place a 4- or 6- toothed comb in place at one end of the electrophoresis tank.
8) Pour 20-12 mL of molten agarose into the tank so that it fills the central cavity and flows under and between the teeth of the comb.
9) Leave the liquid gel to set.
10) Whilst it is setting put two pieces of 42 mm x 22 mm carbon paper at each end of the tank.
11) Pour 10 mL of the buffer solution into the tank so that it covers the surface area of the gel.
12) Gently ease the comb out and place the tank where you will leave it to run.
13) Pipette the loading dye into the wells in the tank. Making note of the DNA you have out into the wells.
14) Put the electrodes at each end of the tank, attaching the crocodile clips to these.
15) Leave to run for several hours. (I stayed after school for and extra hour just to make sure everyone's ran)
16) Disconnect the power supply once finished and remove and dispose of the electrodes.
17) Pour about 10 mL of staining solution over the gel and leave for exactly 4 minutes then return it to the bottle for re-use.
18) Wash the surplus stain from the gel surface with about 5 mL of 70% ethanol for a few seconds, Pour this away nd then rinse with water 3 0r 4 times.
After this the remaining stain will run down the gel. Bands will gradually start appear. The best results, however, appear when the gel is left to develop overnight.
Below are some pictures from mine and Konsti's experiment.
1) Add 100 micro litres of distilled water to a tube of the lambda DNA and mix around using the micro syringe.
2) Cap the tube and flick the bottom of it to mix the contents more.
3) Add 20 micro litres of the lambda DNA to an enzyme filled tube and mix.
4) Repeat this for the other 3 enzyme tubes.
5) Close each tube tightly and place in a holder in an incubator or water bath at 37 degrees celsius for between 30-45 minutes.
6) Melt some agarose gel in hot water. Once completed store in a water bath of between 55-60 degrees celsius until needed.
7) Place a 4- or 6- toothed comb in place at one end of the electrophoresis tank.
8) Pour 20-12 mL of molten agarose into the tank so that it fills the central cavity and flows under and between the teeth of the comb.
9) Leave the liquid gel to set.
10) Whilst it is setting put two pieces of 42 mm x 22 mm carbon paper at each end of the tank.
11) Pour 10 mL of the buffer solution into the tank so that it covers the surface area of the gel.
12) Gently ease the comb out and place the tank where you will leave it to run.
13) Pipette the loading dye into the wells in the tank. Making note of the DNA you have out into the wells.
14) Put the electrodes at each end of the tank, attaching the crocodile clips to these.
15) Leave to run for several hours. (I stayed after school for and extra hour just to make sure everyone's ran)
16) Disconnect the power supply once finished and remove and dispose of the electrodes.
17) Pour about 10 mL of staining solution over the gel and leave for exactly 4 minutes then return it to the bottle for re-use.
18) Wash the surplus stain from the gel surface with about 5 mL of 70% ethanol for a few seconds, Pour this away nd then rinse with water 3 0r 4 times.
After this the remaining stain will run down the gel. Bands will gradually start appear. The best results, however, appear when the gel is left to develop overnight.
Below are some pictures from mine and Konsti's experiment.