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)

  • 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



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