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

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

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

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
NOTE: Results are very likely innaccurate due to the presence of excess primer, dNTPs, buffer and enzyme
 * Used a nanodrop to find the concentration of the PCR products

- Control - 1uL PCR product into 12.5uL diH2O - Positive - 1uL PCR product into 13uL diH2O - Negative - 1uL PCR product into 9.6uL diH2O - 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
 * Performed 2 ligations, one with the recommended 25ng calculated from the nanodrop readings and one with undiluted 5uL PCR product
 * Dilutions:
 * Diluted 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
 * Concentrated Ligations

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:

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.


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