分子生物学之转化技术剖析.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. Imm