User:Daniela A. Garcia S./Notebook/Modeling UNAM-Genomics Mexico/2010/07/29

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=Wet Lab: OmpF/C promoters characterization=

Electrophoresis Gel OmpF PCR_TaqDNApol
In order to probe the PCRs reactions an electroforesis gel was made. This was loadded as follows:
 * Ladder
 * positive control B3K3
 * B3K3 OmpF
 * positive control B1T3
 * B1T3 OmpF



The reactions for B1T3 were sucessful. The next step is to repeat the PCR reaction with the same protocol but this time using the RTTH enzyme.

Oligo Design: GFP suffix FWD
>BBa_E0240 FWD

5'- GCT CTA GAG c TCA CAC AGG AAA GTA CTA GAT GCG -3'


 * Length: 24
 * GC content 45.8%
 * MeltTemp: 56.4ºC

This primer was desinged to amplified an expression plasmid [OmpFp + GFP]

OmpF PCR
PCR1.

Primer forward: Suffix

Primere reverse: Bluepromoter-Preffix

Template: pSB3K3

PCR2.

Primer forward: Suffix

Primere reverse: OmpFpart1-Preffix

Template: pSB3K3

PCR3.

Primer forward: Suffix

Primere reverse: Bluepromoter-Preffix

Template: pSB1T3

PCR4.

Primer forward: Suffix

Primere reverse: OmpFpart1-Preffix

Template: pSB1T3


 * General PCR mixture:
 * 4 microL Template(dilution 1:4)
 * 3 microL Primer forward (5mM)
 * 3 microL Primere reverse (5mM)
 * 6 microL HPLC
 * 6 microL 3.3X XL Buffer
 * 3 microL Mg solution
 * 5 microL dNTP's (0.4 mM)


 * Enzyme(RTTH) mixture for PCR Reaction:
 * 10.5 microL HPLC
 * 9 microL    3.3X XL Buffer
 * 0.5 microL  RTTH


 * Program [35 PCR cycles]
 * 94ºC 5min
 * 94ºC 45seg
 * 60ºC-55ºC 45seg
 * 72ºC 10min
 * 4ºC 3min


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