Search
Search
View additional product information for One Shot™ TOP10 Electrocomp™ E. coli - FAQs (C404052, C404050)
6 product FAQs found
TOP10与TOP10F’细胞的区别仅在于后者包含F’游离体因而带有四环素抗性基因，而且能够从带有f1复制起点的载体菌株中分离单链DNA。F’ 游离体同时还带有lacIq抑制子，因此可用IPTG诱导trc、ta、和lac启动子的表达。TOP10F’细胞需要IPTG诱导进行蓝白斑筛选。
如果插入片段对于宿主细胞有潜在毒性，您可以尝试以下建议：
•转化TOP10或DH5α细胞后，在25-30摄氏度而不是在37摄氏度下孵育。这会降低生长速度并能提高克隆具有潜在毒性的插入片段的几率。
•尝试使用TOP10F’细胞进行转化，但是不在培养板中加入IPTG。这些细胞带lacIq阻抑物抑制从lac启动子起始表达，因此能克隆毒性基因。请注意，没有加入IPTG时不能进行蓝白斑筛选。
•尝试使用Stbl2细胞进行转化。
The only difference between TOP10 and TOP10F' cells is that the latter contain the F' episome that carries the tetracycline resistance gene and allows isolation of single-stranded DNA from vectors that have an f1 origin of replication. The F' episome also carries the lacIq repressor for inducible expression from trc, tac, and lac promoters using IPTG. TOP10F' cells require IPTG induction for blue/white screening.
If the insert is potentially toxic to the host cells, here are some suggestions that you can try:
- After transforming TOP10 or DH5? cells, incubate at 25-30°C instead of 37°C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
- Try using TOP10F' cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
- Try using Stbl2 cells for the transformation.
It is imperative that a cloning strain such as TOP10 be used for characterization of the plasmid, propagation, and maintenance. BL21 cells are wild-type for endA and recA, which could result in poor miniprep quality and a greater chance of plasmid rearrangements due to recombination. In addition, BL21 cells contain the T7 RNA polymerase gene which is expressed at low levels even in the absence of inducer. If the gene is toxic to E. coli, plasmid instability and/or cell death can result.
Yes, TOP10 has the hsdRMS mutation, so this strain can be used to clone DNA from PCR reactions and other non-methylated sources. hsdRMS is a mutation in the system that E. coli uses to recognize foreign DNA. There are two parts to this system, methylation and restriction. E. coli methylate DNA at certain sequences, and if the DNA is not methylated at these sequences it will be recognized as foreign and restricted. Thus, if unmethylated DNA is transformed into E.coli that does not carry the hsdRMS genotype, it is recognized as foreign and enzymatically degraded.