User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/13

AU CHEM-570 Lab Prep

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Bench work Daniel Catt re-ran the 2-stage QuikChange on 6/10/11 with the following modification: first stage had 5 cycles, not 1 Analytical minigel 1.2% agarose gel -- 10μL 1KB ladder -- 5μL QuikChange experimental (Dan's sample from 6/10/11) 5μL QuikChange (-) control (Dan's sample from 6/10/11) -10 -- → ~50' @ 90V → stain with EtBr DpnI digestion add 1μL DpnI to each of Dan's QuikChange reactions → 1h @ 37°C → store @ -20°C Transform DH10B 5μL DpnI-digested QuikChange experimental reaction + 200μL competent DH10B 5μL DpnI-digested QuikChange control reaction + 200μL competent DH10B follow standard transformation protocol plate 200μL → 37°C O/N Results Perfect! Expected a single band at ~6.7 kb, and that's what we got. For this tricky multiple-base insertion, the 2-stage QuikChange with 5 cycles in the 1st stage is the solution.