Index.The use of the microplate reader

From 2013.igem.org

The use of the microplate reader.

1> Firstly according to the steps given above, transform the constructed plasmid into competent cells and clone them by pasting the plates.

2> Add little LB solution and Cam antibiotic solution into the EP tube, then dilute it 30 times or 50 times, and inject them into the 48 orifice, only several orifice are used and add chemicals whose concentrations increase exponentially into the relevant orifice until 500┬Ál. Do not leave out the negative control.

3> Put the plates in the microplate reader, and detect the fluorescence and OD value of them while they are cultured.

4> Set the procedure that detect the amount of the 600nm light absorbed by the bacteria, which is the value of the OD/2, for the area of the cross section of the orifice used by us is roughly around 1cm2, after adding the culture, the depth of the liquid in the orifice turns out to be 0.5cm, and the value we detect is OD/2.

5> The instrument detects the OD value and the amount of the fluorescence every 5 to 10 mins. During this period, the OD value come out first, then the amount of the fluorescence is given. In order that the growth of the bacteria becomes much better, the instrument keeps operating all the time.

6> To detect GFP, the excitation wavelength of the light we use is 485nm, and the emission wavelength is 535nm. To detect CFP1, the excitation wavelength we use is 435nm, and the emission wavelength is 470nm. To detect CFP2, the excitation wavelength if 435nm, and the emission wavelength is 510nm. To detect mCherry, the excitation wavelength is 585nm, and the emission wavelength is 620nm.

7> After measuring overnight, we can get a group of data of the plasmid we are detecting, and then we can draw a figure of it.