a) I have to perform the transformation, for which I selected the TOP 10 strain of E.coli, can i use other strains of E.oli as well?
b) For making competent cells, We incubated the primary culture of E.coli overnight while its secondary culture was incubated for only 3-4 hrs, why?
1) you can use any k12 derivative strain they should transform easy (of course you can you other non-k12 strains but they are more difficult to transform). TOP10 and other cloning strains have mutations that stabilize inserts and enhance the quality of the dna prep (for example endA mutation).
2)the 3-4 hours incubation is to get them to log phase which have a higher transformation efficiency that stationary phase cells.
hi
1- its suitable for transformation DH5alpha , JM109 , JM110
2- for secondary culture 2 hour is enough ( early or middle stage of log phase step in E.coli growth , in this stage bacterial cell wall not very rigid and pore formation occurred in cell wall easier than other stage )
good luck
Muhammad Hassan Instead of Top 10 you can use DH5 alpha and JM109 strains (for maintenance and sequencing purpose) or BL-21 strain (for expression of heterologous protein). These strains are readily available.
About your second questions, Reza Hadavi gave the proper explanation. I would like to add to this that, not all the methods say 3-4 h incubation for 3-4 h, instead to make the ultra-competent cells of E. coli, primary culture is incubated for 6 h (till log phase) and secondary culture is grown o/n at lower temperatures (16-20 C). These cells show higher transformation efficiency. The method is called as : Inoue method for preparation of ultra-competent cells. Refer to Molecular cloning by Sambrook.
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