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
OmpFPCR Taq 290710.jpg









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