工具包是优化与K12的导数和B菌株。全球,一些常用的菌株包括ccdB生存,XL1蓝色和Stbl3。(https://blog.addgene.org/save-time-and-money-by-making-your-own-competent-cells)如果与野生型菌株的细菌,这些菌株通常会有较低的转换效率。因此,我们建议在我们的技巧提高转换效率。

准备在一夜之间文化在磅用颤抖的37°C。——如果从已经主管或electrocompetent细胞,添加1 - 5磅ul细胞5毫升和动摇一夜之间在37°C。——如果从新的殖民地,接种5毫升的磅单殖民地和动摇一夜之间在37°C。

细胞可以在1 x resuspended清洗缓冲和留在冰1½小时转换效率没有明显降低。

细胞可以在1 x resuspended主管缓冲和留在冰1½小时转换效率没有明显降低。

1。使用ZymoBroth而不是哭泣。2。在较低温度下扩大文化ZymoBroth (18 - 30°C, 25 - 26°C实际原因)。较高的温度(即。,33-37 C)可以降低转换效率2 - 10倍。3所示。——mid-log阶段的早期细胞应该收获(OD600nm 0.4 - 0.6)。不超过0.6 OD600nm培养细胞,细胞会减少主管达到后期日志或固定相。4所示。 Once the cells reach an OD between 0.4 and 0.6 and are placed on ice, make sure everything is ice cold during all steps of competent cell preparation. 5. Cool down the centrifuge to 0-4°C before use. 6. Cool down the following items at -20°C prior to use: - Sterile filter pipette tips - Sterile centrifuge bottles or conical tubes to be used for pelleting the cells. - Sterile microcentrifuge tubes to be used for aliquoting (can also be cooled down at -70°C prior to use) - A microcentrifuge tube rack (can also be cooled down at -70°C prior to use) 7. When resuspending the cells: - Do not remove the cells from the ice for too long - Make sure the tubes are in contact with the ice while resuspending - Only remove the cells from the ice for a couple of seconds to check on the progress. 8. Perform a Heat Shock/Outgrowth Procedure using the following protocol: 1. Incubate cells on ice for 5-10 min after addition of plasmid. 2. Incubate cells at 42°C for 45 seconds. 3. Add 450 ml of SOC to the cells. 4. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm. 5. Spread on a pre-warmed culture plate containing the appropriate antibiotic.

是的,缓冲区是非常稳定的,可以储存在室温下几个月。冰箱里把你的装备(4°C)长时间存储。处理前请确保试剂冰冷的收获细胞。

1。整除的细胞的体积转换反应,您希望执行以减少操作的数量。重复冻结/解冻周期会导致一些损失的能力。2。把冷却微型离心机管架的冰或干冰,填满架pre-chilled微型离心机管,整除的细胞,管帽。一旦完成,管道传输到-80°C,供以后使用。在液氮快速冻结并不是必要的。

液态氮仅用于临时冻结前的细胞存储。最常见的方法存储在冰箱里保存-70°C以下。

转化效率取决于几个因素,包括应变、生长条件(如生长介质、温度、通风、细胞密度收获时),质粒(如大小、复制起源、抵抗标记)和长度与质粒的孵化。因此,效率无法保证。