分子生物学实验操作方法与技巧5）课件.ppt

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 ?C until colonies appear. Number of colonies is increased about tenfold by adding 1 ml YPD and shaking for 3 h before spreading thus incubating for a total of 5 h. Expected results: Transformation efficiency up to 2? ?102 transformants/ ?g of DNA. Electroporation protocol for Saccharomyces cerevisiae Growth medium YPD(1% yeast extract,2% bactopeptone,2% dextrose) Washing solution Ice-cold sterile bidistilled water; ice-cold sterile 1M sorbitol. Electroporation solution Ice-cold sterile 1M sorbitol. Cuvette 2mm gap width Making electrocompetent cells 1. Inoculate 500 ml YEPD with an aliquot of an oernight culture or a colony from a plate and grow to an O.D.600 of 1.3-1.5 (about 1? 108 cells/ml). 2.Harvest by centrifugation at 4000g for 5 min at 4 ?C. 3. Wash in 500 ml ice-cold sterile water, centrifuge (4000g,5 min, at 4?C ), repeat washing step with 250 ml water. 4. Resuspend in 20 ml ice-cold sorbitol and centrifuge as above. Resuspend in 0.5ml of 1 M sorbitol to a final volume of 1.0 to 1.5ml, keep on ice. Electroporation of cells 1. Add? 5?l (0.1 ?g )DNA to 65 ?l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Incubate 5 min on ice. Transfer mixture into a prechilled cuvette. 2. Wipe moisture from the cuvette and insert the cuvette into the device. 3. Electroporation: Voltage (V) 1500 V Time constant (?) 5 ms 4. Imme