Skatebro:Digestion

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(Tips)
Current revision (06:07, 9 February 2008) (view source)
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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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