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

Sensor construction hbpR/PhbpC:
We want to amplify these two parts: BBa_E1010 and BBa_B0015.
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).
Solutions were transferred into 1 ml eppendorf tubes and stored at -20°C.
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).

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.

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.

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. 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.

Sensor construction bphR1/bphR2:
A colony after the DH5a transformation was cultured in 3mL of LB + 3µL Amp, at 37°C, 200rpm, overnight

Aug 19