First a little background: Remember that restriction enzymes are proteins and therefore have optimal reaction conditions. All restriction enzymes are stored at -20° C because they are heat labile. But, you will notice that they are not frozen solid; they are stored in 50% glycerol. If there is too much glycerol (>5% v/v) in the reaction, the enzyme will not work properly. Likewise, the salt concentration, pH and temperature must be appropriate for each enzyme. You must do a little research to find the appropriate conditions before you can begin a restriction digestion. To select the proper salt concentration and pH, consult a chart that tells you the optimal reaction conditions; this chart will tell you which buffer to use. You should also verify the optimal temperature for the enzyme(s) you have selected.
1) Decide which buffer you must use and the correct incubation temperature.
2) Decide how much (in microliters) DNA you will digest.
3) Then construct a table similar to the one below:
Do not dispense the liquids onto the sides of the tube. Always
dispense the volumes into the very bottom of the tube!
Suggestion: If you are doing multiple digestions, make a cocktail that contains everything except the DNA and make enough for more tubes than you need. This way when you aliquot the cocktail into the labeled tubes with the DNA, the only variable will be the DNA and it also cuts down on the number of times you have to pipet.
4) As you add each ingredient to a 500
µl microfuge tube, stir it in well with the pipet tip and
NEVER double dip your tip! If caught Double-Dipping, I will call you very early Saturday morning!
5) Make sure all of the liquid is in the bottom of the microfuge tube; spin if necessary.
6) Incubate at the appropriate temperature (typically 37° C) for at least 30 minutes.
If loading onto a gel...
7) When the digestion is completed, add 10X DNA loading dye; one tenth the volume of the digestion.
20 µl digestion reaction
2 µl 10X loading dye
22 µl total volume
The dye will stop the reaction which may be stored at room temperature for a few hours but should be stored at +4° C or colder. Your DNA is now ready to load onto a gel.
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