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

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

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



Personal tools