Skatebro:Digestion
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Tips
- Keep all buffers/enzymes 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
- 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)
- Incubate at 37c for 2 hours (1 minimum and 6 maximum)
- Heat shock for 20 mins at 80c to heat inactivate the enzyme, then store at 4c forever
- Do a purification (elute in 30μL) just like a pcr clean up
- Run a gel to check this step ~before moving on to ligation reaction