IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/12

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pBAD Mutagenesis

Prepare Primers

  • Add 282uL of diH2O to 14.1nMol pBAD reverse primer to make 50uM Basis
 - Make a working solution of 5uM by adding 3.0uL of 50uM primer solution into 27uL of diH2O
  • Add 119uL of diH2O to 11.9nMol pBAD negative forward primer to make 100uM basis
 - Make a working solution of 5uM by adding 1.5uL of 100uM primer solution into 28.5uL of diH2O 
  • Add 115uL of diH2O to 11.5nMol pBAD positive forward primer to make 100uM basis
 - Make a working solution of 5uM by adding 1.5uL of 100uM primer solution into 28.5uL of diH2O

Prepare DNA Template

  • Need 10pg of template per 50uL reaction
  • Dilute 1 uL of 82.04pg/uL solution of DNA template into 4uL of diH20, creating a 16.4pg/uL working solution

Create Master Mix

  • Prepare the Master Mix for 2 50uL(plus 10% overage) reactions using:
  * 22uL 5x Physion HF Buffer - Final concentration 1X
  * 2.2uL of 10mM dNTPs
  * 61.3uL diH2O

Prepare Positive pBAD PCR

  • Add 38.9uL of Master Mix into a 0.2 PCR tube
  • Add 5uL positive forward primer working solution
  • Add 5uL reverse primer working solution
  • Add 0.6uL of working solution of DNA template
  • Just before beginning PCR, add 0.5uL Hot Start DNA Polymerase

Prepare Negative pBAD PCR

  • Add 38.9uL of Master Mix into a 0.2 PCR tube
  • Add 5uL negative forward primer working solution
  • Add 5uL reverse primer working solution
  • Add 0.6uL of working solution of DNA template
  • Just before beginning PCR, add 0.5uL Hot Start DNA Polymerase

Prepare Control Reaction

  • Made according to the Finnzymes Phusion Site Directed Mutagenesis kit

Run PCR

  • Run all three samples using the following temperatures
  • 98°C - 30sec
  • 98°C - 10sec
  • 69.5°C - 30sec
  • 72°C - 60 sec
  • Repeat steps 2-4 25 times
  • 72°C - 10min
  • 4°C hold

Gel Electrophoresis

  • Prepare a standard gel using 1% agarose gel, 0.5 TBE buffer and CYBR green
  • Load 5uL of 1/20 1kb ladder
  • Load 5uL of each PCR product
  • Run at 100V for 1hour

Results

  • High product for control and negative pBAD, very little product for positive pBAD

Ligation

  • Used a nanodrop to find the concentration of the PCR products
' Concentration ng/uL 260/280
Control 311.89 1.3
Positive pBAD 350.32 1.27
Negative pBAD 265.38 1.22

NOTE: Results are very likely innaccurate due to the presence of excess primer, dNTPs, buffer and enzyme

  • Performed 2 ligations, one with the recommended 25ng calculated from the nanodrop readings and one with undiluted 5uL PCR product
  • Dilutions:
 - Control - 1uL PCR product into 12.5uL diH2O            
 - Positive - 1uL PCR product into 13uL diH2O            
 - Negative - 1uL PCR product into 9.6uL diH2O
            
  • Diluted Ligations
- 1uL of diluted sample
- 4uL diH2O
- 5uL of
- 2X Quick Ligation Buffer and mix
- Add 0.5uL of Quick T4 DNA Ligase
- Centrifuge Briefly and incubate at room temperature for 5min
- Chill on ice  
  • Concentrated Ligations
- 5uL PCR product
- 4uL diH2O
- 5uL of
- 2X Quick Ligation Buffer and mix
- Add 0.5uL of Quick T4 DNA Ligase
- Centrifuge Briefly and incubate at room temperature for 5min
- Chill on ice 

Transformation

  • Use 1uL of ligation sample per 100mL competent cells
  • Follow the standard transformation protocol
  • pBAD positive and pBAD negative are plated on LB-AMP
  • Control plasmid is plated on X-gal and IPTG

Measurement of DNA concentration from Heather's miniprep

  • This is a continuation of Heather's stuff from yesterday
  • Used the Finlay Lab nanodrop. Data as shown below:
Sample [DNA] (ng/µL) A260/A280
RBS-RFP-Term 92.06 2.05
Assembly 1 219.41 2.10
P_BAD Reverse 90.87 2.05
pSB1At3 91.70 2.07

Aliquots of Amp and Spread-Plating of Amp-LB

  • Thawed one of the 1 mL portions of 1000X stock into 1.5 mL microfuge tubes
  • Froze the aliquots in the Lagally Lab -20ºC freezer
  • Spread plated the amp (20µL/plate), stored in AMBL 4ºC.