User:Wilfred J. Poppinga/Notebook/Wilfreds Project/2009/08/11

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 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] Metal sensitive promotors protocol #2
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Phosphorylation 100x diluted oligo’s (1 μM)
 * Mix
 * 1 μL PNK
 * 1 μL T4 DNA Ligase buffer
 * 8 μL Oligo’s (FW and REV seperate reactions)
 * Procedure
 * 30 min 37 °C
 * 20 min 65 °C

Annealing
 * Mix
 * 8 μL FW Phosphorylation mix
 * 8 μL REV Phosphorylation mix
 * 4 μL 10 mM NaCl
 * 20 μL MilliQ
 * Procedure
 * Put reactions in (close to) boiling water
 * Let it cool down naturally to ~35 °C

Restriction vector 5 min. 80 °C 15 min. 65 °C
 * Mix
 * 1 μL EcoRI
 * 1 μL XbaI
 * 2 μL Fast digest buffer (10x)
 * 8 μL Vector DNA
 * 8 μL MilliQ
 * Procedure
 * 37 °C, 30 min. incubation
 * Heat inactivation EcoRI and XbaI

Ligation of Metal sensitive promoters in vector 65 °C, 10 min.
 * Mix
 * 2 μL T4 Ligase Buffer
 * 0.5 μL T4 Ligase
 * 1 μL Restriction product (unpurified)
 * 1 μL Annealed oligo’s
 * 18 μL MilliQ
 * Procedure
 * Incubation 16 °C, 1 hour
 * Heat inactivation Ligase

Comments

 * Jolanda's ligation mix was transformed
 * Metal sensitive promotors were ligated
 * -RBS according to paul
 * +RBS & -RBS according to my annealing and Paul's restriction ligation


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