Team:Evry/Notebook/Sensing/PCBs/08-19-2014

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<u>Sensor construction hbpR/PhbpC:</u>
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We want to amplify these two parts: <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> and <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>.
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<b><u>Sensor construction bphR2/PbphR1:</u></b>
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<br/>Corresponding wells were located on 2014 Distribution kit plates and resuspended with 10 µL steril water. That permits to obtain a DNA concentration around 0.2 ng/µl (according to the registry).
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<br/> A colony after the DH5a transformation was cultured in 3mL of LB + 3µL Amp, at 37°C, 200rpm, overnight
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<br/>Solutions were transferred into 1 ml eppendorf tubes and stored at -20°C.
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<br/> A PCR was perform for amplification with the Q5 polymerase and dedicated primers for Golden Gate: 45 and 46 (BBa_E1010) ; 47 and 48 (BBa_B0015).
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<b><u>Survival test on E.coli BL21:</u></b>
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<br/> Bacteria continue to grow for all concentrations of 2 hydroxy-3',4'dichlorobiphenyl.  
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    <center>Table 2: PCR mix Preparation for Q5 amplification.  </center>
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The used program was described on table 1. There was a difference for BBa_B0015, for the 72°C phase time= 15 seconds and no 30.
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    <center>Table 3: IGEM Q5 PCR program thermocycling conditions </center>
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<p align="justify">  Preparation of a 1% agarose gel: 0.56 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X.Microwave 30s by 30s until agarose total dissolution. Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 10µl per sample previously added with 2 µl of loading dye 6X, and 5 µl for ladders. Gel running 45 minutes at 100 mV in TAE 1X buffer. </p>
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    <center>Figure 1: Agarose gel 1%. Lane 1: Purple 2-Log ladder NEB, Lane 2: BBa_E1010 purified PCR product and Lane 3: BBa_B0015 purified PCR product </center>
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We expected to obtain a band for each PCR product. We obtained one band at 700-750 bp for BBa_E1010 PCR product as expected and nothing for BBa_B0015 although we expected a band at 150-200 bp.
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So we decided to perform PCR clean up on BBa_E1010 PCR product with the the GeneJET purification kit (Thermo Scientific). DNA was quantified with the NanoDrop 2000, 37.4 ng/µl with A260/280 = 1.82.
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Latest revision as of 13:10, 6 September 2014

Picture


Sensor construction bphR2/PbphR1:
A colony after the DH5a transformation was cultured in 3mL of LB + 3µL Amp, at 37°C, 200rpm, overnight

Survival test on E.coli BL21:
Bacteria continue to grow for all concentrations of 2 hydroxy-3',4'dichlorobiphenyl.

Aug 19

Retrieved from "http://2014.igem.org/Team:Evry/Notebook/Sensing/PCBs/08-19-2014"