Team:Imperial College London/Project/Arabidopsis/Notebook
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Prior to imaging, roots were washed in PBS to wash off bacteria and facilitate imaging. We imaged the plants incubated with 8ml of bacteria and were able to find bacteria inside one of the roots. A 3D picture was taken of uninfected roots and roots containing bacteria using Z stacking on the confocal micrsocope.<br> | Prior to imaging, roots were washed in PBS to wash off bacteria and facilitate imaging. We imaged the plants incubated with 8ml of bacteria and were able to find bacteria inside one of the roots. A 3D picture was taken of uninfected roots and roots containing bacteria using Z stacking on the confocal micrsocope.<br> | ||
<h1>Tuesday, 9 August 2011</h1> | <h1>Tuesday, 9 August 2011</h1> | ||
- | Si modelled the concentration of auxin secreted of our bacteria to be 10mM. Accordingly, we used concentrations of 10mM, 0.1mM, 0.001mM, 0.01uM, 0.0001uM and 0 to test the effect of different auxin concentrations on the length of the roots and their branching. | + | Si modelled the concentration of auxin secreted of our bacteria to be 10mM. Accordingly, we used concentrations of 10mM, 0.1mM, 0.001mM, 0.01uM, 0.0001uM and 0 to test the effect of different auxin concentrations on the length of the roots and their branching. We made the auxin concentrations by serial dilution and added 10ml of concentrated auxin solution to 100ml of half-MS media each. Twenty-five seeds were added to each flask. The seeds were incubated at 23° and wrapped in aluminium foil to allow the plants to germinate in the dark. They will be allowed to grow in the light in 3 days' time. This follows a protocol described by King et al. (1995). |
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Revision as of 09:14, 10 August 2011
Wednesday, 20 July 2011
Two cultures of wt Arabidopsis thaliana Columbia strain have been set up. Phytogel cultures were set up on Saturday, 15 July and liquid cultures were set up on Wednesday, 20 July.8 of phytogels are seeded with WT arabidopsis at 1 cm below 1 rim of the plate. Each seed is separated to 1 cm. The phytogel is put under light on the shelves in the planting room to allow the space for roots to grow downwards where we grow the roots for 2 weeks.
Wednesday, 27 July 2011
Preparing 5 x 100 ml MS liquid media, autoclaving the media, put in the cold room and preparing the 2 eppendorf of Venus seedling using the described protocolThursday, 28 July 2011
Growing each 20 seeds of Venus in 5 of the prepared 100 ml media by pipetting the seed and transfer the seeds into the media flask under fume cupboard. The flasks are put into the shakers under the light described in the protocolFriday, 29 July 2011
Start modelling the effect of root branching by auxin. Sending the article involving auxin branching to Nina.James field preparing GFP bacteria, as supplied by Dr Tom Ellis's lab
Monday, 1 August 2011
Seedling, preparing media and grow more Venus and GFP in 5 x 2 of 100 ml mediaStocking the GFP bacterial culture in glycerol
Start growing bacterial culture in 188 LB media overnight for plant infection tomorrow
Wednesday, 3 August 2011
Today, we prepared new phytogel cultures as the ones we set up previously were getting too poor in nutrients.We met with Dr Martin Spitaler who advised us on how to prepare samples for the confocal microscopy we will be doing on Friday. The confocal microscopy will focus on imaging GFP expressing bacteria inside Arabidopsis roots to show that uptake of the bacteria takes place. Staining of wt roots with DiD, a lipophilic dye that stains the plant membranes and does not interfere with the absorption or emission spectra of GFP and Dendra, was unsuccessful. However, natural fluorescence was measured in a root in a spectrum that does not interfere with measuring GFP. We should therefore not need to dye the roots before imaging.