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.