旧版pfge脉冲场电泳技术培训4chef故障排除20140224.ppt

Old culture 48 hrs Fresh culture 18 hrs PFGE gel from fresh culture shows no smearing and all bands are distinct. Lysed cells are present in 48 hour old cultures (same organism), characterized by smears on the PFGE gel (blue arrow). Potential problems: Undue stress to bacterial cells prior to casting the plug can cause cell lysis, leading to DNA degradation and is characterized by “smearing” on the gel. Recommendations: Grow cultures on non-selective media Use fresh cultures grown for 14 – 18 hours at appropriate temperature and conditions Do not let cultures sit at room temp for more than an hour before making cell suspensions Do not store cultures at 4C before making cell suspensions Do not use cultures grown for more than 18 hours (contain lysed cells) Do NOT centrifuge or vortex cell suspensions Do NOT leave bacterial cell suspensions at room temp for extended periods of time prior to casting plugs （七） 起始的菌体悬液质量 Preparing cell suspension （八） 制作包埋菌体的小胶块步骤 1% 1.4% 1.2% 1.6% 1.8% S S S When agarose is reheated, water evaporates and the agarose concentration increases, resulting in plugs that do not have the expected agarose percentage. Reheating the agarose 6 times increased the concentration from 1% to 2%. S = standard Potential problems: Agarose that is too hot may damage the cells Agarose thickens and solidifies as it cools, making pipetting difficult and leading to an uneven distribution of cells within plugs Recommendations: Equilibrate melted agarose to 50 – 54oC and keep warm while casting plugs. Limit mechanical force (pipetting) when mixing melted agarose with cell suspensions. Do not re-use plug agarose more than 3 or 4 times. Repeated heating results in loss of fluid and increases the agarose concentration. 反复熔化包埋用琼脂糖 浓度增加 2.0% Exposure time: Too short = difficulty visualizing faint, bottom bands Too long = Increased background, loss of distinction between closely migrating bands Increasing exposure time Under-exposed (6 secs) Just righ