2012分子生物学与生物化学实验操作设计与技能(5).pptVIP

Electroporation of cells 1. Add 10 ?l DNA (5-10 ?g,通常需要通过酶切使质粒线性化) to 80 ?l of electrocompetent cells. Gently mix with a blue-tip pipette several times. Transfer mixture into a prechilled cuvette and incubate on ice for 5 min. 2. Wipe moisture from the cuvette and insert the cuvette into the device. 3. Electroporation: Voltage (V) 1,500 V Time constant (?) 5ms 4. Immediately add 1ml of ice-cold sorbitol to the cuvette. Transfer sample to sterile 15ml tube and incubate for 2 h without shaking at 30 ?C. 5. Spread 200 ?l aliquots on YPDS plates and incubate for 3 days at 30