IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/18

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Plan

  • 1. anneal lox(R) oligos
  • 2. PCR out Cre-LVA
  • 3. restriction digest EcoRI/PsTI for Lox(R) and CreLVA
  • 4. ligate lox(R) and Cre-LVA into two backbones: i) psB1A3, ii) psB1C3
  • 5. transform plasmid

Step 1: Anneal lox(R) oligos

performed according to standard IDT oligo kit instructions.

Step 2: 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

Correct bands were observed, suggesting that the constructs were successful.

Purification of PCR amplified product

using Invitrogen ChargeSwitch kit according to manufacturer's instructions



3. Restriction Digest

using standard UBC iGEM digestion protocol

  • Inserts (cut with EcoRI and SpeI)

Cre-LVA and Lox(R)

  • Backbone cut (cut with PstI and EcoRI)
  • pSB1C3
  • pSB1A3

Incubated reaction mixture for 1hour, 37 degrees Celcius

4. ligation

  • performed using standard UBC iGEM protocol and calculator
  • Ligated (16 degrees Celsius, 2h at PCR machine):'
  • pSB1C3 + LoxR
  • pSB1A3 + LoxR
  • pSB1C3 + Cre-LVA
  • pSB1A3 + Cre-LVA
  • Ligation reaction mixture:

A. For Lox

  • vector: 2.9ul
  • insert: 0.9ul
  • water:4.2ul
  • 10x buffer: 1ul
  • ligase: 1ul

B. For Cre-LVA

  • vector: 2.9ul
  • insert: 2.4ul
  • water:2.7ul
  • 10x buffer: 1ul
  • ligase: 1ul

Transformation

performed using standard UBC iGEM protocol using LB plate with appropriate antibiotics