User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17

Karmella's BioBrick Cloning

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04/17/15 Rene - gRNA plasmids, 5 mL cultures (11 total) Ryan - Receiver plasmids, assembly LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer Ryan - Receiver plasmids, assembly Stage 1 - insert Regulator ORFS Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) Insert ORF(E/X) into Vector (E/X). see Vector plasmid map Stage 2 - insert Promoters Cut & dephos promoters with E/X Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling Phusion PCR AubR, EcoRI F/ XbaI R BjaR, " BraR, " RpaR, " Reagent Volume Mix (x5) Expected: 1. AubR = 851 2. BjaR = 776 3. BraR = 776 4. RpaR = 779 5 μL/lane; 1% agarose; Ladder DNA(plasmid) 0.5 μL --- 10 μM F primer 1.0 5.0 10 μM R primer 1.0 5.0 10 mM dNTPs 1.0 5.0 Phusion Pol. 0.5 2.5 5X HF buffer 10.0 50.0 dH2O 36.0 180.0   50.0 Program: Phusion (block B) 98°C, 3 min 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec] 72°C, 10 min 4°C ∞ Conclusion All four worked. Proceed to next step LCR Development Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer Phusion PCR Reagent Rxn1,2 Expected: 1,2. MV10 = 5191 15 μL/lane; 1% agarose; Ladder DNA(clean linear) 1.0 μL 10 μM F primer 1.0 10 μM R primer 1.0 10 mM dNTPs 1.0 Phusion Pol. 0.5 5X HF buffer 10.0 dH2O 35.5   50.0 Program: Phusion (block A) 98°C, 3 min 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] 72°C, 10 min 4°C ∞ Conclusion Big improvement. Keep this for potential cloning Also try again with higher annealing temp