Team:Paris/August 5

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(→Electrophoresis Purification of PCR)
(→Electrophoresis Purification of PCR)
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''When the PCR cycles were finished,''
''When the PCR cycles were finished,''
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* 10 µL of 6X loading dye were added
+
'''conditions :'''
-
* The samples were then loaded (2 x 30 µL per sample) on a '''1,5% agarose gel'''.
+
 +
* 10µl of ladder 1 kb (unlike 100 pb)
 +
* 2 x 30µl of PCR products added with 10µl of loading Dye 6x
 +
* migration ~30min at 100W on a '''1,5% agarose gel'''.
 +
 +
'''Results of electrophoresis'''
<br>gel 1 [[Image:KR000102.jpg| gel 1|150px]]
<br>gel 1 [[Image:KR000102.jpg| gel 1|150px]]

Revision as of 14:25, 6 August 2008

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Amplification of Promotors of interest (FliA, FliL, FlgA, FlgB, FlhB, FlhDC)

We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.


PCR Protocol

  • Preparation of the templates :--> Resuspend of 1 colony of MG1655 in 100µl of water.


  • List of Oligos :
Number Name Sequence Length Comments
O100 FlgA-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT 58
O101 FlgA-R GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT 58
O102 FlgB-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG 58
O103 FlgB-R GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT 58
O108 FlhB-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG 58
O109 FlhB-R GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC 58
O111 FlhDC-Total-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTCATTTTTGCTTGCTAGCGTACGGAAAA 58 Amplify the both OmpR binding site
O113 FlhDC(nu)-R GTTTCTTCCTGCAGCGGCCGCTACTAGTACAGAATAACCAACTTTATTTTTATG 54 Don't amplify the natural rbs of FlhD (only promoter)
O124 FliL-F GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCAGCGAGAGGCTGTTGGTATTAATGACT 58
O125 FliL-R GTTTCTTCCTGCAGCGGCCGCTACTAGTACCAGCGATGAAATACTTGCCATGCGATTT 58


  • Preparation of PCR mix :

For each samples,


1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)


  • Program PCR: Annealing 55°C - Time élongation 1'30" - Number cycle : 29


Electrophoresis Purification of PCR

When the PCR cycles were finished,

conditions :

  • 10µl of ladder 1 kb (unlike 100 pb)
  • 2 x 30µl of PCR products added with 10µl of loading Dye 6x
  • migration ~30min at 100W on a 1,5% agarose gel.


Results of electrophoresis


gel 1 gel 1 gel 2 gel 2


Name Promotor Gel Band Expected size Measured size
PCR_124 pFlgA 1 2-3 300 pb
PCR_125 pFlgB 1 4-5 300 pb
PCR_126 pFlhB 2 5-6 300 pb
PCR_127 pFlhDC 1 & 2 7 & 2 1.000 pb


==> Remark : for PCR the negative control (templates = water) can be check on the gel n°2, on the band 3-4


==> Conclusion: for the promotors FlgA, FlgB, FlhB we observe the size expected.
We need to repeat the experiments for the promotors FlhDC.


  • After electrophoresis, the bands corresponding to the right amplification were excised and purified using the QIAquick DNA Gel Extraction Kit by "Maurice (QIAcube)".
  • Elution in 50 µL of buffer EB.
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