Hi I am under doctors course, Yong Jae Kim.
I have recently severe problem in homologous recombination for gene integration and knock out into bacterial genome.
I've already check no problem in my experimental procedure with my lab mate.
It's same as conventional way of homologous recombination lambda system.
The strain: K.oxytoca
Comp. cell: early exponential phase, 4 degree celsius
Transfromation: 600 Ohm, 2mm cuvvette, 1.8kV (electroporation)
Refresh: 1hour, fresh LB broth
Spreading for selection: Sm. 70 Cm.30 ug/ml LB plate
Problem: After spreading on the antibiotics selection LB plate and cultivation at 30 degree celsious for 2 days. There are about one hundred colonies found on the plate. However, there is no colony representing positive result in colony pcr which means no integratiton or knock out event occurred at the desire site in the genome.
By the way I have also experienced success in gene integration and knock out by homologous recombination. I found that there are a few colonies below 5 on the selection plate after 2days of cultivation in the success case. Two or three colonies were positive results by colony pcr.
I really have no idea about this phenomenon why in some cases many colonies found which doesn’t show any positive results, and why in successful case a few colonies found which show positive results.
If someone have experienced the same phenomenon with me and solved it, Could you plz let me know????
Sincerely Thank you.
Below 5 positive colonies in total of around 100 colonies, could be the selection plate is problematic:
1) adding antibiotic when the media is still high in temp
2) antibiotic amount is too low
3) selection plate is not prepared fresh
4) bacteria incubate at wrong temp
Thank you for your kind response Vikki Wong.
I've already have successful gene integration under the same LB plate with antibiotics. So The amount of antibiotic seems not to be the main problem.
In addition, In regard to your first response. I have many colonies which are negative results in the experiment with the LB Plate having antibiotics at low temperature.
Because I've check the negative control by the same LB plate and the same incubation temperature, Both fresh state and temperature could not be the main problem........
I sincerely thank you for your help.
but Is there any other recommendation about this problem...... TT
Thank you
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