I was trying to multiply three plasmids (DNA of same ion channel from rat, mouse and human in PCDNA3.1) through transformation.
There were colonies after transformation and sufficient bacteria growth in liquid LB media (with appropriate antibiotics). However, when I try the midiprep only mouse Plasmid DNA prep give any yield. Others give almost no yield.
The rat and muse LB plates had bit different colonies (thicker) and less number of colonies compared to mouse (image attached). However, when the colonies were inoculated in liquid media with antibiotics, there was enough growth of bacteria in all rat, mouse and human samples.
I used 100-150ug/ml Ampicilin in LB plates and LB liquid media. I repeated the experiment three times.
Thanks in Advance :-)
Hi there,
I would check the content of the clones on plates by PCR using plasmid specific primer set before culture for midiprep to make sure of the presence of the expected plasmid.
Hi there,
I would check the content of the clones on plates by PCR using plasmid specific primer set before culture for midiprep to make sure of the presence of the expected plasmid.
On the H and R plates, can you try to grow a few of the large, thicker colonies + a few of the smaller ones (if what I see is indeed smaller colonies)?
And then as Dominique said, you can try to do a quick PCR with a cheap enzyme on either a colony or 0.5-1µL of culture. Then you will be able to prep only the clones with the appropriate plasmid.
Hi,
First things first,You say in your query that you get "almost no yield".I wish to know how much do you get (after quantifying in a biospec) .Second have you tried loading your samples on the gel with your original plasmid ? Also when you say that there was sufficient amount of growth,can you tell us the exact OD of all three cultures that you get.And one more thing,how much amount of LB do you use to inoculate your colonies and for what time do you keep them at 37degrees (overnight/ 3-4 hours)?
Because if its NOT an overnight cultutre chances are that one of the plasmids that you got is high copy number and grows well in that small amount of time whereas the others don't.Try using longer culture times (14-16 hours ) and more culture.For a standard MIDIprep 50ml culture is sufficient.
Thank you all for the informative replies. I'll try the suggestions you made.
Meenakshi Kar-
1) I measured the concentration form Eppendorf Biophotometer. There was no peak in the graph and the concentration was around 0.02g/l (200ul total volume). I usually get around 1g/l total 1ml (depending on the centrifuge I use).
2) No I did not run a gel. I should try it
3)No I did not measure the OD. The flasks were cloudy and not transparent (I usually start midiprep when flasks are cloudy and hazy)
4)I first start a 5 ml starter culture from the colonies. (One colony with 100-150ug/ml Ampicilin). After about 7 hours shaker incubation (37C), I reinoculate 250ul of culture at 250ml flask and shaker incubate for about 18 hours at 37C)
What I can't understand is if the bacteria grow well under Ampicilin, the vector plasmid should be inside the bacteria. But the plasmid does't get extracted during midiprep. I ran a Blank inoculation tube (without putting a colony) with antibiotic added LB media.
I may try another prep (extra midi plus) just to see weather it is working.
Again thank you all for spending your time to help me.
My guess would be that most of the cells don't contain the plasmid anymore.
If your plasmid is instable (often with very large plasmids) and/or the insert is toxic to the cells, it is possible that the cells get rid of the plasmid quickly.
As the ampicilline resistance gene encodes a beta-lactamase, if the cells only express the resistance enzyme at the beginning of the culture, the enzyme is excreted in the media and lowers the antibiotic concentration in the media. Thus, even if cells don't have the plasmid, they can grow, and as E. coli grows very quickly, soon most of the cells don't have the plasmid.
It's similar to the "satellite colonies" phenomenon on ampicilline plates.
To test this hypothesis, 1 ml of the culture can be removed, washed in fresh media (to remove secreted beta-lactamase from the media), diluted and plated on one plate with ampicilline and one plate without antibiotics. If the cells have the plasmid, the number of colonies on both plates should be similar.
If this is indeed the problem, you may try to grow the cells for a shorter time (no more than 16 hours, maybe less), or try with a different antibiotic resistance, or a different plasmid system (to reduce the instability).
Thank you Virginie Dufour. That can be the reason. I used pCDNA3.1 vector. The insert was a Potassium ion channel which is around 5500bp. Therefore the plasmid is fairly big. I will try what you suggest. Thank you again
It is quiet possible that your amp stocks are old because of which in the plates, non AMP resistant bacteria is growing sporadically, and the same can also grow in culture. Maybe you could try using freshly prepared amp stocks just to rule out this possibility.
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