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Team:Valencia/Parts
From 2008.igem.org
Construction of Valencia Team Biobricks
Preparing inserts
Total DNA was extracted from our yeast strains.
UCP-1 was amplified by PCR using oligonucleotides matching the sequence and bearing the appropriate Biobrick prefix and suffix.
Primers' sequences (EcoRI and PstI 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:
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.
