I am facing a weird situation where I got my ligated plasmid in DH5α growing well on the streak plate (overnight), but after I transfered the cells from the streak plate to LB medium, they grow very slowly (overnight shaking 37 degree,200rpm), the cells liquid is still quite clear (eventhough I got some very little pellets after centrifugation). So I used that little pellets for main culture and after 6-8hrs, the solution got turbid. However, I still wonder about the cell quality may affect my plasmid harvest, should I trust the main culture product?. Do you have any opinion about this?, Thank you ~
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* It is not the problem of antibiotics because on streak plate, I used 30ng/ul of Kanamycine whereas in LB only 20ng/ul.
It could still be an antibiotic issue. If you add the Kanamycin when the agar is too hot you won't have as much as you think as some will have broken down. Also Kanamycin can be hard to overcome to begin with if you have a low copy number plasmid that can lead to a longer lag phase in the growth curve.
If you are worried about the quality of your cultures you can always do a quick miniprep from 1 mL before trying the whole lot and seeing how much plasmid you get out followed by a restriction enzyme digest to make sure everything looks ok before doing the full harvest.
It could still be an antibiotic issue. If you add the Kanamycin when the agar is too hot you won't have as much as you think as some will have broken down. Also Kanamycin can be hard to overcome to begin with if you have a low copy number plasmid that can lead to a longer lag phase in the growth curve.
If you are worried about the quality of your cultures you can always do a quick miniprep from 1 mL before trying the whole lot and seeing how much plasmid you get out followed by a restriction enzyme digest to make sure everything looks ok before doing the full harvest.
I used to pick single colonies from the plate into 5-10 ml of culture and grow up until slightly turbid (during the day). I'd then use these seed cultures to inoculate the larger flasks (it was a long time ago so my memories are a bit blurred on this).
If you are growing up from a few colonies into a large volume of LB then I am not surprised it takes a while. Also I used a at least a 5:1 ratio of flask size to media volume to ensure sufficient aeration (i.e. 100 ml LB in 500 ml flask).
As Sarah has suggested just perform a quick mini-prep to see if the bacteria is still contains the plasmid insert .
Sorry I used to grow the single colonies in 5 ml overnight then use that to seed the large volumes (~100 -250 ml) and grow that during the day until the OD at 600 was 1.0, (My memory just returned!).
Thank you Sarah Zilka and Gary Morley for your answers. For overnight culture, I used only 3ml (+Kanamycin), and for main culture I replaced with 5 ml of new LB (+Kanamycin) - just to quickly get the plasmid. I am careful with Kanamicin addition, so it is not the problem of hot LB. However, Yes, I may agree with Sarah...I think there is some affects of Kanamycin on the lag phase. That seems to be the possible reason. I will doublecheck that and get back to you . Thank you ~
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