Skatebro:Digestion

Skatebro:Project08

Tips

• Keep all enzymes and buffers on ice block for the entire process
• Use Dpn1 only if you are cutting a PCR product
• Total volume of 50 μL and ~800ng DNA
• Digest your PCR insert and backbone separately (can also digest a control part in parallel to check that digest is working correctly)
• Incubate at 37c for 2-4 hours and heat shock for 20 mins at 80c
• Don't forget: purification and run a gel of digest before or in parallel to ligation

Steps

1. Add in this order:
   • ?? μL Water (to make 50 μL)
   • 5 μL Buffer 2 (quick vortex first)
   • .5 μL BSA (quick vortex first)
   • ?? μL DNA (whatever neccesary to get 800 ng -- good idea to do a fresh nanodrop of your sample and then make this calculation)
   • .5 μL Pst1 and .5 μL EcoR1 (for example)
   • .5 μL Dpn 1 (only if digesting a PCR product)
2. Incubate at 37c for 2 hours (1 minimum and 6 maximum)
3. Heat shock for 20 mins at 80c to heat inactivate the enzyme, then store at 4c forever
4. Do a purification (elute in 30μL) just like a pcr clean up
5. Run a gel to check this step ~before moving on to ligation reaction