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

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 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] iGEM Project name 1
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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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 Forward (FW): cre-lva fw Reverse (RE): cre-LVA re
 * primer name


 * 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

Cre-LVA and Lox(R)
 * Inserts (cut with EcoRI and SpeI)


 * 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

A. For Lox
 * Ligation reaction mixture:
 * 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


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