IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/10/04
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Repeated experiment: Working on LovTAP.Reporter systems: PCR to fuse trpL promoter
Due to the previous PCR reactionsamplified two bands around the expected size. I'm going to test new temperature conditions and template dilutions, as well I will make some reactions using Dimethyl Sulfoxide.
1.Insert the trpL promoter through a PCR reaction to the plasmid pSB1C3.
-trpL promoter sequence
New Primer designed
-Primer_trpL_reverse (5'->3'): NheI site + trpL promoter + XbaI site + EcoRI site TTGCTAGCGTGAACTTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGCCTCTAGAAGCGGCCGCGAATTC
-Primer forward (5'->3'): Suffix
-Template: Plasmid pSB1C3
The reaction starts at 95°C during 5 min.
Each cycle is programmed as follows:
After the cycle 35, the temperature changes to 72°C during 5 min and ends at 4°C.
Results:PCR with promoter trpL