Restriction digest of pBR322 with EcoRI

 

Materials:

Methods:

1. assuming DNA in TE buffer ( because added the gel loading dye already by mistake), set up a digest for pBR322 cut with EcoRI in a 1.5ml eppendorf tube, by first pipetting the following:

   

2. Add restriction nuclease EcoRI and mix the contents well by gently tapping the bottom of the tube with finger (avoid bubbles). There is one EcoRI cut site on pBR322.
3. optinally: briefly spin down the contents of the tube in centrifuge.
4. incubate for 1.0 - 1.5 hours
5. inactivate the restriction enzyme with 10 min

Results: ( 1/27/99)

Performed the digest on the pBR322 miniprepre sample from previous step. Because added gel loading dye by accident, used twich as much restriction nuclease EcoRI than needed. Heat inactivated for 15 min because also inactivated HindIII at same time.