Tianjin/FLIP-FLOP/More details

Solid LB culture 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.

The fragment usually is gotten from gel purification, sometimes from nucleic acid co-depositing.
Reagents

Calculating
The amount of vector and insert fragment needed for a successful ligation is decided by formula given by openwetware.

Reaction
Ligate reaction is taken by TaKaRa DNA Ligation Kit Ver.2.0. Total reaction volume is 10μL, one half of which is DNA solution and another half is Solution I containing T4 ligation enzyme. Typical reaction temperature is 16°C. Reaction time varies from 45 minutes to 6 hours depending on the specific condition.

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.

Open the bottle of medium and flame the mouth, measure out 2 ml to fill 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 fluorometry. AHL is detected by the detector used in Bio-Diode.