IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/07/28

iGEM Project name 1

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Making Lock/Key/Control into Ampicillin and Chloramphenicol construction plasmids (pSB1A3 and pSB1C3) 1. PCR 2. Digestion 3. Ligation 1. PCR performed using standard UBC iGEM PCR protocol primers Forward (FW): G1004 Reverse (RE): G1005 reagents for each reaction (μL) 10x reaction buffer: 2.5 10μM Forward primer: 1.25 10μM Reverse primer: 1.25 10mM dNTP: 0.5 sdH20: 82.72 liquid culture: 2.2 (transferred using autoclaved wooden stick) Taq: 0.88 PCR steps [temperature | time ] Initial denaturation: 94°C | 120s Denaturation: 94°C | 30s Annealing: 64°C | 30s Extension: 72°C | 15s Final extension: 72°C | 45s Step 2-4 repeated 30 cycles PCR result The negative control (water added to reaction mixture instead of DNA) seems to be contaminated As the contaminant was amplified using the G1004/1005 primers, the construct would contain the BB cut sites (thus cannot be used for restriction digest) A prior PCR purified Lock/Key/Control dsDNA from July 16 (currently in the 4°C fridge). Will restriction digest these again and clone them into Amp and Chlor backbones. 2. Restriction Digest using standard UBC iGEM digestion protocol Inserts (cut with EcoRI and SpeI) Lock/Key/Control dsDNA from July 16 Backbone cut (cut with PstI and EcoRI) pSB1A3 pSB1C3 Incubated reaction mixture for 1hour, 37 degrees Celcius Heat inactivation was performed after incubation to inactivate enzymes 3. Ligation 4. ligation performed using standard UBC iGEM protocol and calculator Ligated (16 degrees Celsius, 1h at PCR machine): pSB1C3 + Lock pSB1A3 + Lock pSB1C3 + Key pSB1A3 + Key pSB1C3 + Control pSB1A3 + Control Ligation reaction mixture: vector: 3.4μL insert: 3.0μL water: 11.6μL 10x buffer: 1μL ligase: 1μL Transformed 10μL of each ligation into DH5α using standard UBC iGEM protocols cPCR of ASEM 6(A), 6(B), 6(C) to verfy constructs performed using standard UBC iGEM cPCR protocol (cultures were in triplicate) primers Forward (FW): VF2 Reverse (RE): VR2 reagents for each reaction (μL) 10x reaction buffer: 2.5 10μM Forward primer: 1.25 10μM Reverse primer: 1.25 10mM dNTP: 0.5 Taq polymerase: 0.5 sdH20: 18.8 liquid culture: 1.0 (transferred using autoclaved wooden stick) PCR steps [temperature | time ] Initial denaturation: 94°C | 120s Denaturation: 94°C | 30s Annealing: 56°C | 30s Extension: 72°C | 30s Final extension: 72°C | 90s Step 2-4 repeated 30 cycles Gel electrophoresis to verify cPCR bands no water contamination (good). Throw out G1004 and G1005 primers as they must be contaminated. expected band size: 366bp. 6A and 6C displayed correct bands. 6A and 6C were miniprepped using standard UBC iGEM protocols and diluted to 100μg/μL GFP Controls 1. obtained from Matt 2 types: GFP-LVa and bright GFP 2. incubated at 37°C for 24h (took cells out from incubator on July 29