IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/10/03

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Repeated experiment: Working on LovTAP.Reporter systems: PCR to fuse trpL promoter
Due to the primer with the trpL promoter was designed by Zepeda without taking into account that the promoter region had two SpeI sites, thus being wrong the usage of an SpeI site in front of it, I had to design a new primer changing the SpeI site by an NheI site.

Experimental Setup
1.Insert the trpL promoter through a PCR reaction to the plasmid pSB1C3.

-trpL promoter sequence

tggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagtacgcAagttcacgtaaaaagggtat

NewcPrimer designed

-Primer_trpL_reverse (5'->3'): NheI site + trpL promoter + XbaI site + EcoRI site TTGCTAGCGTGAACTTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGCCTCTAGAAGCGGCCGCGAATTC

-Primer forward (5'->3'): Suffix

-Template: Plasmid pSB1C3


 * PCR taq platinum reaction mixture


 * 35 PCR programmed cycles:

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



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