Skatebro:Digestion: Difference between revisions
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*Digest your PCR insert and backbone separately (can also digest a control part in parallel to check that digest is working correctly) | *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 | *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 | *Don't forget!: do a purification and run a gel of digest before or in parallel to ligation | ||
==Steps== | ==Steps== | ||
#Add in | #Add in order: (50μL little pcr-sized tube) | ||
#*#*Enough water to bring the total volume to 50μL | |||
#*5 μL Buffer 2 (quick vortex first) | #*5 μL Buffer 2 (quick vortex first) | ||
#*.5 μL BSA (quick vortex first) | #*.5 μL BSA (quick vortex first) |
Revision as of 20:46, 31 January 2008
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!: do a purification and run a gel of digest before or in parallel to ligation
Steps
- Add in order: (50μL little pcr-sized tube)
- Enough water to bring the total volume to 50μL
- 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