User:Daniela A. Garcia S./Notebook/Modeling UNAM-Genomics Mexico/2010/08/17

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=Wet Lab: OmpC/F promoter=

OmpC digestion
The OmpCp is inserted in the plasmid pSB1A2 between the EcoRI and XbaI restriction enzymes. It is done a digestion process to validate the two sites for the restriction enzymes. Total 30μL
 * DNA 20μL
 * BSA 0.3μL
 * Buffer 4 3μL
 * EcoRI enzyme 1μL
 * XbaI enzyme 1μL
 * HPLC ---4.7μL

Overnigth reaction @ 37ºC

OmpF RTTH PCR
PCR1 Template: PCR4(2010/08/05)
 * FWD Suffix
 * REV OmpF (2)

PCR2 Template: PCR5(2010/08/05)
 * FWD Preffix
 * REV Suffix


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


 * Enzyme(RTTH) mixture for PCR Reaction:
 * 21 μL HPLC
 * 18 μL    3.3X XL Buffer
 * 1 μL  RTTH
 * 20 μL for each reaction


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

Hold


 * C2R1S1
 * C3R1S3


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