Team:Valencia/Parts

From 2009.igem.org

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===Preparing inserts===
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Total DNA was extracted from our yeast strains.<br>
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AEQ was amplified by PCR using oligonucleotides matching the sequence and bearing the appropriate Biobrick prefix and suffix.<br>
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Primers' sequences (EcoRI and XbaI sites in bold):<br>
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Forward: 5' '''gAATTC'''gCggCCgCTTCTAgATggTgAgTTCgACAACTTC 3';<br>   
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Reverse: 5' TACTAgTAgCggCCg'''CTgCAg'''CTATgTggTgCAgTCCACTg 3' <br>
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PCR was conducted as follows:<br>
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<ol>A first denaturation cycle
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<ol>94º 3'</ol>
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Followed by 30 amplification cycles:
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<ol>94º 30''
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60º 1' 30''
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72º 1'</ol>
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And a final extension step:
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<ol>72º 10'</ol>
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</ol>
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'''Results:'''<br>
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[[Image:.jpg]]<br>
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Intense amplicons, of the expected size, (about 1 kb) were produced for UCP-1, 175-deleted and 76 deleted. <br>
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Amplicons were digested (H buffer) with EcoRI y PstI.<br>

Revision as of 14:53, 3 October 2009















Preparing inserts

Total DNA was extracted from our yeast strains.
AEQ was amplified by PCR using oligonucleotides matching the sequence and bearing the appropriate Biobrick prefix and suffix.


Primers' sequences (EcoRI and XbaI sites in bold):
Forward: 5' gAATTCgCggCCgCTTCTAgATggTgAgTTCgACAACTTC 3';
Reverse: 5' TACTAgTAgCggCCgCTgCAgCTATgTggTgCAgTCCACTg 3'


PCR was conducted as follows:

    A first denaturation cycle
      94º 3'

    Followed by 30 amplification cycles:

      94º 30 60º 1' 30 72º 1'

    And a final extension step:

      72º 10'


Results:

File:.jpg
Intense amplicons, of the expected size, (about 1 kb) were produced for UCP-1, 175-deleted and 76 deleted.
Amplicons were digested (H buffer) with EcoRI y PstI.

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