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Team:Valencia/Parts
From 2009.igem.org
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.
