DNA Digest

 

Stock solutions

                                    RNaseA (20mg/ml)

 

If frozen, thaw the DNA preparation plate and keep on ice. 

 

Make a cocktail mixture of the following reagents:

                                                            For 1 reaction              For 100 reactions

            10X Reaction buffer                      1ml                                  100ml

            EcoRI                                           10Units                          1000Units          

            RNaseA (20mg/ml)                     0.05ml                                 5ml

            dH2O                                       Q.S. to 5ml                       Q.S. to 500ml

 

Mix these reagents thoroughly avoiding bubbles in the mixture keeping all reagents on ice throughout the prep. 

 

Aliquot 5ml into each well of a 96-well plate for PCR machines (Bio-Rad 2239441) or 0.2ml strip tubes for PCR (MbP 3418) and place on ice.  

 

Add 5ml of the DNA  to the cocktail mixture, but do not draw up and down to mix.  This will sheer the DNA.  Tap the plates on the benchtop or a quick spin in the centrifuge to make sure all liquid is at the bottom of the tube.   Seal the DNA plate  with a plate sealer (Fisher 14-245-192B) when done

 

Cover the plates with a plate cover (Applied Biosystems N801-0550) or strip caps for the strip tubes and place in a PCR thermocycler.  Set the thermocycler for 2 hours at 37°C and then 5°C for ¥. 

 

When the run is complete, take the samples out of the thermocycler and add 2ml of 6X DNA gel loading dye to each sample.

 

The samples are now ready to load on to a gel, or can be placed with covers on inside of a Ziploc back for storage at 4°C