User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/28

Karmella's BioBrick Cloning

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04/28/15 Ryan - minipreps & digests Ryan - PCR & Dig/Lig (promoter insert trial #2) Minipreps Use Sigma GenElute; elute with 75 μL elution solution BraR/MRV colonies - picked 4 additional colonies from original cloning plate Cut w/ E/X Bra/MRV 3 Bra/MRV 4 Bra/MRV 5 Bra/MRV 6 promoter/Regulator/MRV colonies Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut) pAub/AubR/MRV pBja/BjaR/MRV pRpa/RpaR/MRV Reagent Volume Expected: 1-4. Bra/MRV = ~3200, 776 Empty MRV E/X = ~3200 5. pAub/AubR/MRV = 4240 6. pBja/BjaR/MRV = 4099 7. pRpa/RpaR/MRV = 4116 Empty vector = ~4000, 971 15 μL/lane; 1% agarose; Ladder DNA(plasmid) 3.0 μL 10X buffer 1.5 enzyme 1 1.0 enzyme 2 1.0 dH2O 8.5   15 μL --> 37°C/ ~15 min. Measure [DNA] Sample OD 260 260/280 ng/μL BraR/MRV #5 0.465 1.913 465.4 Conclusions BraR/MRV - Success! Keep colony 5 (lane 3) promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts Ryan - PCR & Dig/Lig (promoter insert trial #2) Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters) 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 PCR-amplify promoter inserts (same primers as Stage 1) Cut & dephos promoters with E/X Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling Assemblies pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087 pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977 pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977 pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980 Measure conc.'s Values from last week, except BraR/MRV, which was measured today Sample OD260 260/280 ng/μL 1. AubR/MRV 0.258 1.906 257.7 2. BjaR/MRV 0.177 1.904 177.3 3. BraR/MRV #5 0.465 1.913 465.4 4. RpaR/MRV 0.246 1.896 246.2 PCR-amplify promoters 2x 50 μL rxns each pAubR, EcoRI fwd, XbaI rev pBjaR, EcoRI fwd, XbaI rev pBraR, EcoRI fwd, XbaI rev pRpaR, EcoRI fwd, XbaI rev Reagent Vol Mix (x2) Expected: 1. pAubR = 153 2. pBjaR = 122 3. pBraR = 214 4. pRpaR = 136 Hover name 5 μL/lane; 1% agarose; Ladder gBlock DNA (2 ng/μL) 0.5 1.0 10 μM XbaI rev 1.0 2.0 10 μM EcoRI fwd 1.0 2.0 2x GoTaq green 25.0 50.0 dH2O 22.5 8.7   50.0 μL Thermal cycler: Labnet - GOTAQ 95°C 3 min. 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec] 72°C 3 min. 4°C, ∞