User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/28

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
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Objective
Troubleshoot ligation (see notes from last week) Prep media, if needed, for ER2566 competent cells (just make and use fresh once DNA is ready)
 * transform old ligation into commercial cells
 * re-ligate O/N @ 16°C
 * start double-digest again

Bench work

 * 1) Double-digest
 * &rarr; 2h @ 37°C
 * &rarr; 30' @ 65°C
 * 20' is sufficient
 * &rarr; store insert reaction @ -20°C


 * 1) Phosphatase vectors
 * 2) * add 1μL Alkaline Phosphatase to double-digest of pTXB1His and pTXB1 from step #1
 * 3) *&rarr; 15' @ 37°C
 * 4) *&rarr; store @ -20°C
 * 5) Transformation into NovaBlue
 * 6) * Re-transform ligation from last week into Matt Hartings' commercially competent NovaBlue E. coli cells
 * 7) 5μL pTXB1His+BSA ligation + 50μL cells
 * 8) 5μL pTXB1+BSA ligation + 50μL cells
 * 9) * Followed standard transformation protocol for each sample in step #1
 * 10) *&rarr; heat shock step was 45"
 * 11) *&rarr; plated 100μL each on LBAmp and stored the rest @ 4°C
 * 12) **&rarr; 37°C O/N
 * Kathryn Muratore 15:49, 29 June 2011 (EDT): I forgot to do the O/N ligation @ 16°C!


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