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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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
 * 1) Analytical minigel
 * 2) * 1.2% agarose gel
 * 3) 10μL 1KB ladder
 * 4) 5μL QuikChange experimental (Dan's sample from 6/10/11)
 * 5) 5μL QuikChange (-) control (Dan's sample from 6/10/11)
 * 6) -10 --
 * 7) *&rarr; ~50' @ 90V
 * 8) *&rarr; stain with EtBr
 * 9) DpnI digestion
 * 10) * add 1μL DpnI to each of Dan's QuikChange reactions
 * 11) *&rarr; 1h @ 37°C
 * 12) *&rarr; store @ -20°C
 * 13)  Transform DH10B
 * 14) 5μL DpnI-digested QuikChange experimental reaction + 200μL  competent DH10B
 * 15) 5μL DpnI-digested QuikChange control reaction + 200μL  competent DH10B
 * 16) * follow standard transformation protocol
 * 17) * plate 200μL
 * 18) *&rarr; 37°C O/N
 * 1) * plate 200μL
 * 2) *&rarr; 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.


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