IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/06

iGEM Project name 1

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cPCR of ASEM 16 to ASEM 21(5 assemblies) performed using standard UBC iGEM cPCR protocol (cultures for ASEM 16 - 21 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 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 | 72s Final extension: 72°C | 216s Step 2-4 repeated 30 cycles Gel electrophoresis of ASEM 16 to ASEM 21 cPCR samples triplicates were labeled a,b,c ASEM 16: Expected band size: 405 Conclusion: actual band sizes too large ASEM 17: Expected band size: 1179 Conclusion: actual band sizes too large; 17c was correct -> miniprep * ASEM 18 Expected band size: 1100 Conclusion: 18a and c sizes correct -> miniprep the brighter band ASEM 19 Expected band size: 1137 Conclusion: correct band size for 19a and b-> miniprep ASEM 20 Expected band size: 1178 Conclusion: correct band size for 20b-> miniprep ASEM 21 Expected band size: 371 Conclusion: correct band size for 21b water control: no band -> good, since it's the negative control ASEM1(C), 12(C), control-lock, key 1 sent for sequencing Miniprep of ASEM 17, 18, 19, 20, and 21 Using RAf's Alkaline Lysis protocol Sample ID | A260 | A280| 250/280 ASEM 17 | 44.895 | 22.389 | 2.01 ASEM 18 | 48.223 | 23.256 | 2.07 ASEM 19 | 40.835 | 20.418 | 2.00 ASEM 20 | 39.314 | 19.067 | 2.06 ASEM 21 | 46.664 | 23.009 | 2.03 Diluted all miniprep to 100μg/μL Addition of LVA tag to Cre through PCR Expected length: 1.2 kb primer name Forward (FW): cre-lva fw Reverse (RE): cre-LVA re reagents for each reaction 10x reaction buffer: 2.5 10μM Forward primer: 1.25 10μM Reverse primer: 1.25 10mM dNTP: 0.5 sdH20: 0.2 DNA: 0.5 PCR steps [temperature | time ] Initial denaturation: 94°C | 30s Denaturation: 94°C | 30s Annealing: 68°C | 30s Extension: 72°C | 1min12s Final extension: 72°C | 3min36s Step 2-4 repeated 30 cycles Gel electrophoresis of PCRed Cre-LVA successful Purification of PCR amplified product using Invitrogen ChargeSwitch kit according to manufacturer's instructions DNA stored at -20°C Annealing [lox] (reversed) 1. add 40μL of RNase-free Duplex Buffer to each of the stands to make 100μM stocks 2. add 90μL of RNase-free Duplex Buffer to 10μL of 100μM stocks to make 10μM working solutions 3. add 50μL of each working solution together in a PCR tube; put into PCR machine (heat up to 95°C, ramp down to 24°C at 3% ramp rate) Assembly (2, 9, 10, 11, 13, 27, 28, and lox into Amp construction plasmid pSB1A3) 1. Digestion, 1h at 37°C using standard UBC iGEM digestion protocol Prefix samples (cut with EcoRI and SpeI) BBa_J23100 °C Lock 1 (Amp) BBa_I13453 Suffix samples (cut with XbaI and PstI) BBa_J31001 BBa_E0034 BBa_E1010 BBa_E0022 BBa_K145015 Backbone cut (cut with PstI and EcoRI) pSB1C3 2. 3A assembly ligation into pSB1A3, 16°C BBa_K145015 and BBa_J31001 were inserted into the backbone BBa_K145015 ligation into plasmid Suffix Part: 8.79ul 10x Buffer: 2ul Ligase: 1ul Vector: 4ul Water: 2.99ul BBa_J31001 ligation into plasmid Suffix Part: 7.09ul 10x Buffer: 2ul Ligase: 1ul Vector: 4ul Water: 4.69ul To be transformed by Amelia 3. Putting Cre-LVA and lox into Amp plasmid (pSB1A3) 1. digest Cre-LVA and pSB1A3 with EcoR1 and Pst1 2. ligation Ligation reaction mixture: A. For Lox vector: 7.2ul insert: 1.0ul water:3.3ul 10x buffer: 2ul ligase: 1ul B. For Cre-LVA vector: 3.0ul insert: 30.0ul water:11.0ul 10x buffer: 5ul ligase: 1ul To be transformed by Amelia