Successful fill-in plasmid (G56-1) amplification

Materials

SOC medium 400μl
Amp Agar plates 2* plates
Inoculating loop
Successful fill-in plasmid:PB5 - HS4 - 5xUAS - mCherrynuc - 2A - Bla - loxN - GpA - HS4 - PB3

Methods

1.Place the tubes on ice, allow cell to thaw on ice.
2.Divide the competent cells into 2 tubes,50μL each tube, Mark them with G1, G2 .
3.Add 2μL plasmid to each tube, shaking slightly, keep the bacteria in ice at 4°C for 45min.
4.Put the samples at 42°C for 90s to transfer the plasmids into the cells.
5.Immediately transfer the samples to ice. And keep the samples in ice for 2 min.
6.Transfer each one of the samples to 0.2 ml of liquid SOC media for 45 min at 37°C，200 rpm shaker.
7.Centrifuge the samples 4000 rpm, 5min.
8.Discard the supernatant liquid.Add 50μL samples to amp LB agar plant respectively and distribute the bacteria in plate.
9.Incubate the plates at 37°C .

10.Incubate the plates at 4°C on 13:00 next day.
11.Pick up a monoclone (an isolated large colony) on the plate with white pipette tip .
12.Drop the pipette tip directly into the tube which contains 6ml LB medium with antibiotics.
13.Incubate LB medium tubes, 200rmp, 37C, 14 hour