Team:Colombia/Interlab

Methods

Strains and culture

All transformations were carried out in E. coli TOP10. This bacterium was cultured in LB (suplemented with the appropiate antibiotic for each case) at 37 °C.

Transformations and plasmidic DNA extration

This process was carried out as it is indicated in our protocols page.

Fluorescence Microscopy

All the pictures shown here were taken using a Eclipse-Ti Nikon® ECLIPSE Ti inverted microscope equiped with an ANDOR iXon Ultra 897 camera. The filter used was FITC.

Fluorescence Microscopy

The fluorometric measures were taken using a DGEASGAES. The measures were carried out three times while 10 min with an ON culture diluted in a 1:10 proportion in 2mL of fresh LB, with a previous measuring of OD.

Image Analysis

The image analysis were performed with ImageJ 1.46r using Nikon ND Reader and interactive 3D Surface Plot pluggins. We used Analyzing fluorescence microscopy images with ImageJ (Queen's University Belfast 2014) as guide. You can download this useful material here.

Validation

The following gel shows respectively the plasmids with the devices and the restriction profiles for each of the tree devices digested with EcoRI and PstI. For restrictions we obtained two bands (the plasmid and the device) with the expected sizes in all cases.

GELES :)

You can check the size of each device with the next table:

Measures

Fluorescence microscopy

Here are the images of the transformed cells with this device:

Light (left) and FITC (right)

And the analysis for relative fluorescence among them performed from the last image above:

Relative fluorescence among cells

Fluorometry

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