We used the Student's guide of the Lambda protocol from the National Centre for Biotechnology Education to cut the DNA of the bacteriophage lambda. We did this with three different restriction enzymes that all cut the DNA strand after a special DNA sequence. The following enzymes were used in our attempt: HindIII, BamHI and EcoRI. We also had a fourth container which DNA where we didn't use any restriction enzymes to control the others.
First of all, you need the DNA of the bacteriophage lambda. It should be mixed with a little bit destilled water so that it is fluid. Then you should put with a microsyringe 20µL of the DNA solution into each of the tubes which contain the enzymes and into the one without any enzyme. Watch out that you use a fresh tip every time to prevent the DNA from mixing. Close those tubs then and put them into a 37 degrees warm water bath for 30 up to 45 minutes. Put the into a foam holder during this time to prevent them from drowning.
During this time you should start to prepare the agarose gel. Use boiling water to melt and melt the gel in it until it is stired. Keep it at a temperature of 55 until 60 degrees until you use it. Take an elctrophoresis tank and put the gel into it. Leave it for the next 20-30 minutes and place a 4-toothed comb at one end. Put between 10 and 12 ml into the tank and make sure that everywhere is equally much. Place carbon fibre tissue which fills out completely both ends of the tank and then leave it like this to dry.
Put enough TBE buffer solution into the tank that it flood into both ends where the electrodes are and covers the gel to a depth of 2-3 mm. Then take 2µL into a microsyringe and mix it with one DNA sample. Put it into one of the left over holes of the comb in the gel and repeat this for every different DNA sample. Watch out where you put which DNA or write it down. Put on each side of the container a crocodile clip which is connected to a 36 volt transformer. The carbon fibre tissue has to touch the solution. Then leave it like this for around two hours.
After this time you should remove the electrodes and our of the buffer solution. Put around 10ml staining slution on the top of the gel leave it for 4 minutes. Add 5ml of a 70% ethanol solution and leave it for a few seconds. Wash the surace with alittle bit water and you will be able to see the strands of DNA when you hold it against a bright background. Now you can check your pattern of cut DNA strands with the usual pattern.
First of all, you need the DNA of the bacteriophage lambda. It should be mixed with a little bit destilled water so that it is fluid. Then you should put with a microsyringe 20µL of the DNA solution into each of the tubes which contain the enzymes and into the one without any enzyme. Watch out that you use a fresh tip every time to prevent the DNA from mixing. Close those tubs then and put them into a 37 degrees warm water bath for 30 up to 45 minutes. Put the into a foam holder during this time to prevent them from drowning.
During this time you should start to prepare the agarose gel. Use boiling water to melt and melt the gel in it until it is stired. Keep it at a temperature of 55 until 60 degrees until you use it. Take an elctrophoresis tank and put the gel into it. Leave it for the next 20-30 minutes and place a 4-toothed comb at one end. Put between 10 and 12 ml into the tank and make sure that everywhere is equally much. Place carbon fibre tissue which fills out completely both ends of the tank and then leave it like this to dry.
Put enough TBE buffer solution into the tank that it flood into both ends where the electrodes are and covers the gel to a depth of 2-3 mm. Then take 2µL into a microsyringe and mix it with one DNA sample. Put it into one of the left over holes of the comb in the gel and repeat this for every different DNA sample. Watch out where you put which DNA or write it down. Put on each side of the container a crocodile clip which is connected to a 36 volt transformer. The carbon fibre tissue has to touch the solution. Then leave it like this for around two hours.
After this time you should remove the electrodes and our of the buffer solution. Put around 10ml staining slution on the top of the gel leave it for 4 minutes. Add 5ml of a 70% ethanol solution and leave it for a few seconds. Wash the surace with alittle bit water and you will be able to see the strands of DNA when you hold it against a bright background. Now you can check your pattern of cut DNA strands with the usual pattern.