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| - | [[Skatebro:Project08]]
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| - | ==Tips==
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| - | *Keep all buffers/enzymes on ice block for the entire process
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| - | *Do calculations based on fresh dna spec reading
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| - | *50 μL total volume
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| - | *need ~800ng DNA to cut!! (yes thats a lot)
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| - | *Digest your PCR insert and backbone separately
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| - | *Use Dpn1 ''only'' if you are cutting a PCR product
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| - | *Incubate at 37c for 2-4 hours and heat shock for 20 mins at 80c
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| - | *Don't forget!: do a purification and run a gel of digest ~before ligation
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| - | ==Controls==
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| - | *Digest a control part in parallel to check that digest is working correctly
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| - | *Run cut, cleaned-up dna on a gel
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| - | ==Steps==
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| - | #Add in order: (50μL little pcr-sized tube)
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| - | #*?? μL water to bring the total volume to 50μL
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| - | #*5 μL Buffer 2 (quick vortex first)
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| - | #*.5 μL BSA (quick vortex first)
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| - | #*?? μL DNA (whatever neccesary to get 800 ng -- good idea to do a fresh nanodrop of your sample and then make this calculation)
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| - | #*.5 μL Pst1 and .5 μL EcoR1 (for example)
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| - | #*.5 μL Dpn 1 (''only'' if digesting a PCR product)
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| - | #Incubate at 37c for 2 hours (1 minimum and 6 maximum)
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| - | #Heat shock for 20 mins at 80c to heat inactivate the enzyme, then store at 4c forever
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| - | #Do a purification (elute in 30μL) just like a pcr clean up
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| - | #Run a gel to check this step ~before moving on to ligation reaction
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