User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/22

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12/22/09 ✓ Cultures: KAH120-125 in MV2 (2 each); KAH135 (2) ✓ Digests/ dephos.: Vectors for Strept apt into JDS42 & 43 ✓ Kinase treatment + oligo annealing: Strept apt Assemblies Strep apt/JDS42: Strept apt/(NotI dsoligo)/50 + (1) JDS42/(NotI)/7468 Strep apt/JDS43: Strept apt/(NotI dsoligo)/50 + (2) JDS43/(NotI)/7478 > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) 10.0 10x buffer 3.0 NotI 2.0 dH2O 15.0   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. JDS42(NotI) 0.053 2.08 5.6 2. JDS43(NotI) 0.139 3.24 6.9 > Dephosphorylation (Roche) Reagent Volume DNA (clean digest) up to 17 μL (~100 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~5 ng/μL Kinase treatment & oligo annealing Joe's: Strep apt S1 F NotI + Strep apt S1 R NotI > Kinase treatment DNA (oligos, 100 μM) up to 23 μL (3 μL ea.) 10 x PNK (polynucleotide kinase) buffer 3.0 10mM ATP 2.0 T4 polynucleotide kinase (PNK) 2.0 dH2O ---   30 μL --> 37°C/ 1.5 hours --> Add 4 uL 500 mM NaCl --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight in water bath