User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/22

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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

 * 1) Ligation
 * 2) * Insert = gel-purified double-digested BSA PCR from yesterday
 * 3) * Vector and Vector(His) = gel-purified double-digested pTXB1 or pTXB1His, respectively, from yesterday
 * 4) 5μL Insert + 10μL Vector(His) + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
 * 5) 5μL Insert + 10μL Vector + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
 * 6) *&rarr; 20' @ 37°C
 * 7) *&rarr; 15' @ 65°C
 * 8) * Note that I should have done a control reaction (vector without insert)
 * 9) * Kathryn Muratore 13:55, 24 June 2011 (EDT): Also note that I had 8 times more DNA in the reaction than intended (see yesterday's results)
 * 10) Transform DH10B
 * 11) Followed standard  transformation protocol for each sample in step #1
 * 12) *&rarr; heat shock step was 1'20" (intended to do 1' flat)
 * 13) *&rarr; plated 200μL each on LBAmp and stored the rest @ 4°C
 * 14) **&rarr; 37°C O/N
 * 15) Streak ER2566
 * 16) Streaked out cell stock from NEB onto LB
 * 17) *&rarr; 37°C O/N


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