Team:Evry/Notebook/Sensing/PCBs/week8

From 2014.igem.org

Revision as of 16:53, 20 August 2014 by JulieZ (Talk | contribs)

Week 8

Sensing/PCBs

08.18.2014

Sensor construction hbpR/PhbpC
IMAGE
Symbol
Table 1: Received Primer Sensor construction hbpR/hbpC

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).
IMAGE
Symbol
Table 2: PCR mix Preparation for Q5 amplification.
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.
IMAGE
Symbol
Table 3: IGEM Q5 PCR program thermocycling conditions

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.

IMAGE
Symbol
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
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. 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.

08.20.2014

Sensor construction hbpR2/PhbpR1
We received sequencing reads: that match perfectly to the registry sequence.
Sensor construction hbpR/PhbpC
The amplification by PCR was performed another time to amplify BBa_B0015 with overhangs. Protocol was the same as 08/19/2014. To verify PCR products, 10 µl was loaded on a 1% agarose gel with 2 µl of loading dye 6X. A really slight band was visible between 200 and 300 bp. It was to weak to obtain a picture with the camera. According to the ladder scale, the band correspond to 20-30 ng, so approximately 2-3 ng/µl of DNA for the PCR product.

08.21.2014

08.22.2014

08.23.2014

08.24.2014

Retrieved from "http://2014.igem.org/Team:Evry/Notebook/Sensing/PCBs/week8"