User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/06

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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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Objective
Improve transformation efficiency

Bench work

 * 1) Transform NovaBlue
 * 2) 2.5μL V(His)+I ligation from  last week + 25μL NovaBlue
 * 3) 2.5μL V(His) neg ctrl ligation from  last week + 25μL NovaBlue
 * 4) * follow transformation protocol
 * 5) * heat shock is 80s @ 42°C
 * 6) * add 450μL SOC for recovery
 * 7) *&rarr; plate 100μL on LBAmp100
 * 8) *&rarr; 37°C O/N
 * 9) Re-plate transformants
 * 10) * spread 450μL of DH10B transformants from yesterday on LBAmp100
 * 11) *&rarr; 37°C O/N
 * 12) Electrocompetent DH10B - starter culture
 * 13) * 3 mL LB + 50μL E. coli DH10B from thawed chemically-competent cells
 * 14) *&rarr; 37°C O/N w/ shaking
 * 15) Culture ER2566
 * 16) * for glycerol stock and also to confirm that media is okay since there have been problems with some of my students growing starter cultures.
 * 17) * 3 mL LB + colony E. coli ER2566 from streaked plate
 * 18) *&rarr; 37°C O/N w/ shaking
 * 19) Test Amp100
 * 20) * #* 3 mL LB + DH10B/pKSeAATHis colony #1 from one of Tamra's transformation tests this week x 2
 * 21) *&rarr; 37°C O/N w/ shaking

Results

 * No growth on yesterday's plates


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