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===Sequential digestion using HindIII/EcoRI on the Stoffel DNA and on pUC19=== *Reaction 1: No enzyme control 2.0 uL pUC19/Stoffel (~2ug) 3.0 uL EcoRI buffer (10x) 25.0 uL Nuclease-free water *Reaction 2: Single HindIII 2.0 uL pUC19/Stoffel (~2ug) 3.0 uL HindIII buffer (10x) 3.0 uL HindIII 22.0 uL Nuclease-free water *Reaction 3: Single EcoRI 2.0 uL pUC19/Stoffel (~2ug) 3.0 uL EcoRI buffer (10x) 3.0 uL EcoRI 22.0 uL Nuclease-free water *Incubate each reaction at 37C for 1 hour. *Reaction 4: Double negative 3.5 uL HindIII buffer (10x) 15.0 uL Reaction 1 33.5 uL Nuclease-free water *Reaction 5: Double, HindIII then EcoRI 1.5 uL EcoRI 3.5 uL EcoRI buffer (10x) 15.0 uL Reaction 2 30.0 uL Nuclease-free water *Reaction 6: Double, EcoRI then HindIII 1.5 uL HindIII 3.5 uL HindIII buffer (10x) 15.0 uL Reaction 3 30.0 uL Nuclease-free water *Incubate reactions 4,5,6 at 37C for 1 hour. *Run digestion on 1% agarose gel for 1 hour at 50 V. To verify the degree of completion for double digest. Double digestion of pUC19 is required since undigested/single digest vectors will generate colonies under ampicillin selection. *Purify double digest products using 50% volume of AmPure Beads. * Agarose gel: (pUC19 digestion only) ** The gel may be slanted since the double digest product is higher than the single-digest product. [[File:Stoffel_production_pUC19_digestion.png | 200px]]
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