Team:Evry/Notebook/Sensing/PCBs/08-20-2014

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<u>Sensor construction hbpR2/PhbpR1</u>
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<u>Sensor construction bphR2/PbphR1</u>
<br>We received sequencing reads: that match perfectly to the registry sequence.
<br>We received sequencing reads: that match perfectly to the registry sequence.
<br>26DJ54
<br>26DJ54
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TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA
TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA
TGATAATAATGGTTTCTTAGA
TGATAATAATGGTTTCTTAGA
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<br/>
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<br>
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<br/>
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<br><u>Sensor construction hbpR/PhbpC</u>
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<br/> Plasmid with bphR2 gene was purified with NucleoSpin Plamsid protocol (Macherey-Nagel) => we obtained 227,5ng/µL
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<br>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.
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<br/>This plasmid was digested according this protocol:
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<ol>
 +
<li>Add:
 +
<ul>
 +
<li> sterilized water: qsp 20µL
 +
<li> template DNA : 500ng
 +
<li> buffer 2.1: 2µL
 +
<li> BSA: 0,2µL
 +
<li> EcoRI: 1µL
 +
<li> PstI: 1µL
 +
</ul>
 +
<li>Reverse for mix
 +
<li>Incubate at 37°C during 45mn
 +
<li>Incubate at 80°C during 20mn
 +
<li> Ligation was not possible because pSB1C3 plasmid was not digested so the digested plasmid (bphR2 gene) was stored at -20°C
 +
</ol>
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<br/>
 +
<u>Survival test on E.coli BL21:</u>
 +
<br/>Bacteria survive again for different concentrations but they were very concentrated
 +
<br/>
 +
<br/> A dilution of the medium has been done for the positive control and the six different concentrations:<br/>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2014/3/3a/2nd_Survival_test_1st_day.jpg" alt="image not found" /></div>
</p>
</p>

Latest revision as of 23:08, 17 October 2014

Picture

Sensor construction bphR2/PbphR1
We received sequencing reads: that match perfectly to the registry sequence.
26DJ54
AATAGGCGTTATCACGAGGCAGAANTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGATGTCCCTGG GTGACATGCGTGATTTGGCCGCCACGCGGATCGCGCTCGAGAGCGAAGCGTTACGCCAAAGCGTGCTGAATGGTGACGCTGAATGGGAGGCGCGGATCGTCAGTTC GTTTCACCGACTGTCATTGATTGAAGAGCCCACGATGCGGGATCCGGCTCGCTGGTTTAATGAGTGGGAGCCAGTCAACCGCGGTTTTCACGAAGCTCTTATCTCT GCCTGTTCGTCCGTCTGGATCCGGCGGTTCCTGTCCATCCTGTATGTGCATATGGAGCGCTACCGCCGATTGACTGCTTACTAGTAGCGGCCGCTGCAGTCCGGCA AAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC GGCTGCGGCGAGCGGTATCA GCTCACTCAGGG

26DJ55
CGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGC AGCGGCCGCTACTAGTAAGCAGTCAATCGGCGGTAGCGCTCCATATGCACATACAGGATGGACAGGAACCGCCGGATCCAGACGGACGAACAGGCAGAGATAAGAG CTTCGTGAAAACCGCGGTTGACTGGCTCCCACTCATTAAACCAGCGAGCCGGATCCCGCATCGTGGGCTCTTCAATCAATGACAGTCGGTGAAACGAACTGACGAT CCGCGCCTCCCATTCAGCGTCACCATTCAGCACGCTTTGGCGTAACGCTTCGCTCTCGAGCGCGATCCGCGTGGCGGCCAAATCACGCATGTCACCCAGGGACATC TCTAGAAGCGGCCGCGAATTCCAGAAATCATCCTTAGCGAAAGCTAAGGATTTTTTTTATCTGAAATTCTGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA TGATAATAATGGTTTCTTAGA


Plasmid with bphR2 gene was purified with NucleoSpin Plamsid protocol (Macherey-Nagel) => we obtained 227,5ng/µL
This plasmid was digested according this protocol:

  1. Add:
    • sterilized water: qsp 20µL
    • template DNA : 500ng
    • buffer 2.1: 2µL
    • BSA: 0,2µL
    • EcoRI: 1µL
    • PstI: 1µL
  2. Reverse for mix
  3. Incubate at 37°C during 45mn
  4. Incubate at 80°C during 20mn
  5. Ligation was not possible because pSB1C3 plasmid was not digested so the digested plasmid (bphR2 gene) was stored at -20°C

Survival test on E.coli BL21:
Bacteria survive again for different concentrations but they were very concentrated

A dilution of the medium has been done for the positive control and the six different concentrations:
image not found

Aug 20

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