how long to culture the bacteria after electroporation - (Aug/17/2006 )
after electrotransportion, i will culture the competent cells for 1 hour or 40mins,but i don't know how to decide the time of culture and how long is suitable? THX in advance! 
I shake the cells anywhere between 40 - 60 min depending on if I have time or not. It really doesnt matter.
it is said shaking can promote the efficiency of electrotransporton.but what is the reason? and how long is suitable?
I think after electroporation, the cells r quite vulnerable and shaking can help them to sort of help recovery and grow. As the cells will die if plated immediately. The shaking is basically to let them recover in optimum media, thats y one doesnt add anibiotics in the SOB/SOC media used to add after electroporation.
yes, i agree with your point,and i think the plasmid carries antibiotic gene in general,culturing the cells after electroporation will make the antibiotic gene express, subsequencingly,the cell can survive when they are plated,do you think so ? more important, i wonder how long the antibiotic gene can express by shaking?
yes, i agree with your point,and i think the plasmid carries antibiotic gene in general,culturing the cells after electroporation will make the antibiotic gene express, subsequencingly,the cell can survive when they are plated,do you think so ? more important, i wonder how long the antibiotic gene can express by shaking?
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that's right. it is to let the bacteria recover in a rich medium and express antibiotic resistance gene in order to be able to survive in medium with antibiotics when plated.
i don't know the minimum time required but we shake it at least 40 min.
please read page 4 of this pdf its great.
http://www.edgebio.com/tech/faq/EB5Alpha_F...ansformation%22
E.coli, have very much faster growth rates and therefore higher oxygen demands (up to 800 nmol L-1 h-1) and therefore need more oxygen mass transfer than can be achieved through diffusion or rocking. To obtain high oxygen mass transfer rates, vigorously shaken flasks can be used which yield oxygen transfer rates of 15-30 nmol L-1 h-1 . The downside is that such vigorous agitation, particularly with a stirred system, will shear delicate cells.
For mammalian cell cultures, this is not too tough a challenge since their oxygen requirements are very low (0.3-80 nmol L-1 h-1) and can be meet through passive diffusion for small volume cultures in flats or in rocked systems in which the oxygen transfer rate is less than 1 nmol oxygen L-1 h-1 — just about meeting oxygen demands at lower cell densities.
hope this will help.
quite right, since prior to electroporation, the bugs were at -70. the antibiotic genes must express before plating. and shaking is not critical. exposure to 37c is. i do it for 45 m in a water bath following electroporation, and then plate on an amp+ medium.
- viv