Second Attempt: Restriction Digest of pGFP(LVA)

 

Materials:

Methods:

1. thaw pGFP(LVA) in vector form (from Clontech)
2. set up a two digests for pGFP(LVA) cut with HindIII in a 1.5ml eppendorf tube, by first pipetting the following into two seperate tubes:

   

3. perform two dilutions of HindIII: and and pipet each into separate tubes (here diluted with   and pipetted , but can also try pipetting instead of diluting)
4. mix the contents well by gently tapping the bottom of the tube with finger (avoid bubbles). HindIII cuts pGFP(LVA) at two MCS sites (positions 249 and 1062).
5. briefly spin down the contents of the tube in centrifuge.
6. incubate for 1.0 - 1.5 hours
7. the result should be two double-stranded DNA parts from the original pGFP(LVA) of 3339 bases: GFP(LVA) insert (base positions: 249-1062, total: 813 bases), rest of plasmid (total: 2526 bases).
8. inactivate the restriction enzyme with 15 min

Results: ( 1/29/99)
Performed 2 digests as specified above, but also by accident performed a digest by adding HindIII (not diluted). Will run all 3 samples on separation gel.