传统噬菌体制剂的一个主要限制是滴度的可变性,盐,和细菌污染物之间的连续传播。在这里,我们介绍了用于快速有效制备均质噬菌体(噬菌体)股票的噬菌体(PoT)协议。这种方法产生均匀的,实验室规模,高滴度(高达10(10-11)PFU·ml(-1)),内毒素减少的噬菌体库,可用于消除噬菌体繁殖之间的变异性并改善噬菌体的分子特征。该方法包括五个主要部分,包括噬菌体繁殖,通过0.22μm过滤和氯仿处理清除噬菌体,通过超滤浓缩噬菌体,内毒素去除,以及用于连续实验室使用的噬菌体库的制备和存储。从>100mL的起始液体裂解物中,PoT协议产生了一个干净的,同质,实验室噬菌体库,在短短两天内噬菌体回收效率达到85%。相比之下,传统方法需要五天以上才能产生高滴度,但较低体积的噬菌体原液,回收效率仅为4%。噬菌体库可以进一步纯化以去除细菌内毒素,减少内毒素浓度超过3,000倍,同时保持噬菌体滴度。PoT协议专注于类似T的噬菌体,但广泛适用于可以繁殖到足够滴度的各种噬菌体,生产同质的,适用于分子和细胞测定的高滴度噬菌体库。
A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 10(10-11) PFU·ml(-1)), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.