We have transformed positive test control insert (given with pET-32 Xa/LIC cloning kit) with E. coli DH5 alpha competent cells. After 24hours of incubation on LB plate (40 µg/ml ampicillin containing medium) at 37°C, bacterial lawn was found, instead of colony. What may be reason behind this lawn formation?
(Image of LB plate is attached)
Any comments or suggestions will be greatly appreciated. Thank you in advance.
It looks like the ampicillin is distributed unevenly in your plate and is not at an inhibitory concentration.
Try using 100 ug/ml of ampicillin final concentration. Add concentrate to molten agar and mix well before pouring.
It occurs probably because you did not spread the mixture evenly or the plate was wet before you wrapped and incubate at 37 degrees. Try to spread the mixture well and ensure that the plate doesnot contain any mixture in liquid form. You may also reduce the volume of the mixture.
Thank you ma'am.
Mixture means inoculation of our transformed cells?
Are you suggesting reducing the amount of inoculation during plating?
This looks to me that the plate was very wet and when cells started growing they were "washed and spread out" from the liquid on the plate from the condensation. if the ampicillin was too low or uneven you usually find the max concentration in the center and the edges of the plate are less. But you do have some discrete colonies at one side.
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