I have transformed DH5@ with 2 ul of an old stock of pET15b and pET28a through heat shock method. Competent cell was prepared through method described by Inoue et al. Heat shock was performed for 45 seconds at 42 oC and after 2 minutes incubation in ice, 1 ml SOC media was added. Culture was incubated at 37 oC for one hour with shaking at 220 rpm. After one hour, 200 ul was spread on LB agar plates supplemented with 100 ug/ml and 50 ug/ml ampicillin and kanamycin respectively. Colonies are appearing after 16 hours. pET15b has higher colonies than pET28a. Is it a normal growth or their is some thing wrong because doubling time for E. coli is only 20 minutes.
I do not see any problem with your protocol. 16 h to growth is fine.
Bacterial plates usually take overnight to form proper colonies. However, it also depends on the number of cells, plate conditions such as antibiotics and so on. In your case it is normal.
I always use overnight (±16 hours) incubation after transformation. It is fine, and I think standard procedure at most labs. If I clone a large plasmid, like a BAC, I reduce temperature (30 degrees) and increase incubation time.
Basically, if you obtain colonies your transformation most likely worked. Just always use colony PCR or restriction assay to confirm your insert/plasmid.
Good luck.
Usually the incubation time is overnight, but it is normal to see colonies after 16 hours.
I do not see any problem with your protocol. 16 h to growth is fine.
- Agree with the above
- Actual growth rate will depend on the insert as some inserts are inimical to bacterial mitosis and therefore the cells remain in a prolonged lag phase
- Irrespective, as mentioned, if you put them in the incubator over night for 12-16 hours you should alway see colonies; If you dont you need to repeat
16-18 h incubation is fine. The number of colonies you obtain, sometimes vary according to the plasmid and insert (if any) used, happened to me but it did not affect the required outcome. Good luck.
As recommended above usual time for proper colony formation for plasmid transformed in DH5 alpha competent cells is overnight (±16 hours) with amp or kan selection.In case of ampicillin selection formation of satellite colonies along with the actual colonies is possible if the incubation time is extended beyond the recommended time.Also the heat shock time can be extended up-to 90 secs for better transformation.
the protocol what you are following is perfect and 16 hours incubation is fine enough to get good colonies.
if you use Ampicillin antibiotic for selection of single colonies for your experiment there might be chances for satellite colonies formation.
@ Sandeep Kumar ,
Appearance of colonies after up to 16 hours is generally not too bad, but the relatively slow growth may be due to a number of factors. However, if every other things look good in your subsequent steps, you probably need not worry about the duration it took considering that it might be influenced by the nature of your insert. On the other hand, you may want to take care of the following possibilities to handle your fear:
- Low transformation efficiency resulting from handling and all...
- Be sure that the concentration of the two antibiotics are not mistakenly way above what you intended...
- Take all recommended precautions during transformation...
Hi Sandeep,
you are following exactly the right protocol of transformation. you are also getting good results because this is a usual growing time of E. coli. If you are cloning some gene in it then also confirm your results via colony PCR and sequencing so you become confident about your gene transformation in DH5alpha.
If you further want to improve it so you can increase the time of heat shock to 60 seconds, and time of incubation on ice after heat shock to 10 minutes and you can also increase the concentration of antibiotics for best results.
Wish you all the best for your future experiments:)
usually overnight incubation is fine...... if ampicillin plate is not having any colonies, then keep the plate at room temperature you will get some statelite colonies
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