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Name: Bal
Status: Student
Grade:  Other
Location: AL
Country: United States
Date: December 2005


Question:
In gene cloning experiments what would happen if transformed E.coli cells were not incubated in LB broth before being plated on agar plate?

Also what would happen if they were incubated in LB btoh for an extended time before being plated?



Replies:
Selection for transformed cells is based on antibiotic resistance. The inserted DNA contains a gene that confers antibiotic resistance, and the cells are plated on agar plates containing an antibiotic. This is important because only a small fraction of the cells actually takes up the foreign DNA. The Luria Broth (LB) step allows the cells to recover from the shock of the transformation (either chemicals or electrical charges are used to shock the cells so their membranes become more permeable to the DNA you are trying to insert). The LB step also allows the cells to begin to express the genes on the transformed plasmid, including the antibiotic resistance gene. So, you get a higher number of tranformants on the plate if you allow the cells to recover in LB for about an hour.

If you allow the cells to incubate for an extended period of time in LB, they will start to divide. Since there is no antibiotic in the broth, all the cells multiply, including those without transformed DNA. Since there is no selection for cells containing the foreign DNA (plasmid), there is no "incentive" for the cells to retain the plasmid. Over time, the percentage of transformed cells will become increasingly smaller.

Bottom line: I wouldn't be too concerned about letting the cells go a couple of hours in LB (if I couldn't get them plated after one hour), but I wouldn't let them grow overnight before plating them.

Paul Mahoney, PhD


I have found that the longer the cells are in the LB broth, the better the recovery, ie. the more transformed colonies there are. This isn't necessarily because more cells were transformed, but the ones that ARE have a chance to multiply. It depends on what your goals of the experiment are. If you just want to show that transformation is possible and to get a qualitative result, I recommend letting the cells sit in LB for up to 4 hours and then plating them. This may require that you do this for your students after they are gone. I have even been known to OVERPLATE what they did in class with a few drops of a 4 hour culture. I have also found that I get better results if I let the plates incubate for at least 48 hours. I try to schedule this lab for a Friday. If you want to do a quantitative transformation, ie. have them calculate the transformation efficiency (how many cells were actually transformed), then you should at least allow the cells to recover in LB for as long as you can in the time you have. I think a critical step in transformation in my experience is when the kids add the cells to the calcium chloride in the first place-they usually just drop the cells in in a "hunk". This only allows the cells on the outside of the hunk to be exposed to the plasmid. If they emulsify, or break up, the hunk of cells so that the liquid looks cloudy, then more of the cells will be exposed and you have a better chance of some of the cells being transformed.

Good luck

vanhoeck


If the transformation mixture is plated directly onto selective agar media (plates containing antibiotic), fewer transformed colonies will appear per mL plated. Growing the transformed cells in LB broth will simply increase the number of transformed cells per mL.

Ron Baker, Ph.D.



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