Lab_protocols分子生物学实验方法技术汇总讲述.docVIP

LABORATORY PROTOCOLS Institute of Cell Biology and Genetics [1] General PCR Protocol Here in this Lab, we do 25μl PCR reactions in 0.25ml microfuge tubes. You can do PCR in different size reaction volumes and in smaller tubes as long as they fit in the thermocycler. PCR is very sensitive to contamination from outside DNAs. Steps should be taken to reduce the chance for contamination, such as wearing gloves, and not spitting in the tubes. Assemble your reactions on ice. 实验前准备：PCR开始前，将SDW, 10× Buffer, 10mM dNTPs, 10μM primers, 模板，MgCl2取出放在冰上。依次加入上述反应物，加完后，再加酶(酶要放在冰上)。酶用完后马上放回-20°C保存。 1. Gently vortex (涡流) and briefly centrifuge all solutions after thawing (融化，解冻). 2. Add, in a thin-walled PCR tube, on ice. SDW to 25μl Buffer (10X): 2.5μl dNTPs (10 mM): 1μl primers (10 μM): 1μl template: Xμl MgCl2 (25 mM): 1.5μl Taq polymerase (keep at -20 °C) 0.25μl 3. Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube. 4. Place samples in a thermocycler and start PCR. General PCR cycyles are as follows: 1 cycle-4 mins-94°C; 35 cycles-30 sec-94°C; 30 sec-X°C*; X sec-72°C; 1 cycle-10 mins-72°C. [2] PCR product purification protocol (TaKaRa Agarose Gel DNA Purification Kit Ver.2.0) 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Cut the gel into small pieces. 2. Weigh the gel slice in a colorless tube. Add 3-5 volumes (1% agarose, 3 volumes; 1-1.5%, 4; 1.5-2%, 5) of DR-I Buffer to 1 volume of gel (100 mg ~ 100 μl). 3. Mix thoroughly. Incubate at 75°C for 6-10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. 4. After the gel slice has dissolved completely, add 0.5 DR-I Buffer volume of DR-II to the sample and mix. If DNA f