Hello friends,
I wanted to know in which case there is a better probability (practically not theoritically) to get more plasmid yield?
A) Pickup a transformed colony and put it for 12-16 hrs and then isolate DNA?
B) Pickup a transformed colony and put it for 12-16 hrs followed 2-3 hrs secondary culture and then isolate DNA?
Thanks
Jitendra
Hi, I never tried to incubate my bacteria culture the next day for 2-3hr again if I want to isolate plasmid. It was never necessary to do this because I got enough material. From a mini isolation I got minimum 2µg but mostly around 6-7.5 µg which was enough for sequencing and resctriction digests. Also for a midi an over night culture of 12-16hr was enough. Only if I wanted to make a maxi or a mega, I made a preculture over the day and used this to start my over night culture.
If you make a over night culture and then a second culture you should have in mind that your bacterias from the over night culture already in the final phase in the plateau phase and if you want to start a culture again it takes time before the bacteria are again in the proliferative phase. In general the second protocol is used to make competent cells. After this 2-3hr your cells are in the proliferative phase with an OD around 0.3-0.4 which is optimal to make competent cells.
Hi. If you extract plasmid with use of too old bacterial culture because of increases in dead bacterial you can't obtain highest plasmid amount. so you need to determine the best time for pick up the bacteria from culture that depends on amount of medium, shake rate and initial inoculation.
best regards
Hi, I never tried to incubate my bacteria culture the next day for 2-3hr again if I want to isolate plasmid. It was never necessary to do this because I got enough material. From a mini isolation I got minimum 2µg but mostly around 6-7.5 µg which was enough for sequencing and resctriction digests. Also for a midi an over night culture of 12-16hr was enough. Only if I wanted to make a maxi or a mega, I made a preculture over the day and used this to start my over night culture.
If you make a over night culture and then a second culture you should have in mind that your bacterias from the over night culture already in the final phase in the plateau phase and if you want to start a culture again it takes time before the bacteria are again in the proliferative phase. In general the second protocol is used to make competent cells. After this 2-3hr your cells are in the proliferative phase with an OD around 0.3-0.4 which is optimal to make competent cells.
Your overnight cultures will be fine for making your plasmid preps. While Mohsen is correct that if the cultures are too old then the yield may go down, but 12-16 hours is not too old.
Hello,
I agree with Michael, O/N culture is ok, I usually do that and I never had problems.
Hi
What we usualy do when we need to do a midi from a single colony is make a pre-inoculum. we pick up a colony from the plate and culture it in 5ml of LB during the day and in the end of the day we use those 5ml to inoculate a larger amount of LB to culture overnight. Usualy this is enough and we get a nice amount of plasmidic dna.
hope it was of help
Dear Pedro and Monica thanks for your answer
Monica, Thanks for including a different approach to culture.
regards
Jiten
Hi,
Its always better to isolate plasmids from the secondary culture when the cells are healthy and in log phase. It is observed that better yield is obtained throgh this method.
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