Tianjin/FLIP-FLOP/More details
From 2007.igem.org
Experiment | ||
Solid LB medium is used for agar plate. For a typical liquid culture, 5 ml or 100ml of appropriate medium is used, and condition is supposed to be 37°C and 220rpm. The amount of time needs to wait depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. To get the transformation colony, waiting about 12-16 hours. Using plasmid extracting kit we get the plasmid DNA for two purposes. One is to verify the plasmid using enzyme digesting and the other is purifying the fragment using enzyme digesting and agarose gel electrophoresis. | ||
Colony PCR can be used to screen colonies containing desired plasmid.But it always brings out the incorrect result, so under most conditions we use enzyme digesting to verify the desired palsmid.
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The fragment usually is gotten from gel purification, sometimes from nucleic acid co-depositing. Calculating Reaction | ||
Using CaCl2 solution to make competent cells, and foreign DNA is transformed chemically to competent cells. If foreign DNA is plasmid, using 2-3 μL to transform 100 μL competent cells. If it is ligation product, transform the total 10 μL product to 100 μL competent cells. Opening the bottle of medium and flame the mouth, bringing 2 ml to 10 ml Eppendorf Tube.Centrifugate the tube and pipette in 5 ml of the suspension to another tube for detecting the extracellular AHL.Then the deposit is used to detect GFP using fluorometer. The concentration of AHL is detected by the detector used in Bio-Diode. |