User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30

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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
Continue trying to get BSA-intein construct cloned.

Bench work

 * 1) Ligation
 * 2) * Using new digested DNA
 * 3) ** Previous purification could have been problematic, but hopefully the yield is higher and there is less ethanol this time.
 * 4) * Insert: gel-purified NheI/SapI digested BSA PCR from yesterday
 * 5) * Vector(His): gel-purified NheI/SapI/Phosphatased pTXB1His1 from yesterday
 * 6) * Vector: gel-purified NheI/SapI/Phosphatased pTXB1 from yesterday
 * 7) 8.5μL Insert + 8.5μL Vector(His) + 2μL buffer + 1μL T4 DNA Ligase
 * 8) 8.5μL Insert + 8.5μL Vector + 2μL buffer + 1μL T4 DNA Ligase
 * 9) 8.5μL Vector(His) + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control)
 * 10) 8.5μL Vector + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control)
 * 11) *&rarr; 16°C O/N
 * 12) *&rarr; store @ -20°C
 * 13) Transformation
 * 14) * Tranform yesterday's O/N ligation into DH10B
 * 15) * Follow standard transformation protocol except:
 * 16) 100μL DH10B + 5μL V(His)+I from  yesterday
 * 17) 100μL DH10B + 5μL V+I from  yesterday
 * 18) 100μL DH10B + 5μL V(His) neg ctrl from  yesterday
 * 19) 100μL DH10B + 5μL V neg ctrl from  yesterday
 * 20) * heat shock time is 45s
 * 21) * plate 100μL each
 * 22) Re-plate  yesterday's NovaBlue transformation
 * 23) * 450μL of each re-plated
 * 24) *&rarr; 37°C O/N


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