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

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= Making Lock/Key/Control into Ampicillin and Chloramphenicol construction plasmids (pSB1A3 and pSB1C3)=
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 * 1. PCR
 * 2. Digestion
 * 3. Ligation

1. PCR

 * performed using standard UBC iGEM PCR protocol

Forward (FW): G1004 Reverse (RE): G1005
 * primers


 * 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

Lock/Key/Control dsDNA from July 16
 * Inserts (cut with EcoRI and SpeI)


 * 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

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)

Forward (FW): VF2 Reverse (RE): VR2
 * primers


 * 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


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