I have read other questions about kanamycin, but didn't answer my question. I'm trying to expand a plasmid containing an mammalian-derived Zn transporter. The plasmid also carries a Kanamycin-resistance gene. I have transformed E. coli DH5alpha by heat shock at 42 C. The first problem is that the colonies that I get on the agar plate are smaller than usual, even after 20 h. After selecting a colony, I tried growing in both LB media and SOC media with Kanamycin (10 ug/ml) overnight. The OD is only slightly lower than other flasks grown in parallel (same E. coli with pcDNA3.1-gfp and Ampicillin). The plasmid yield from midiprep is substantially lower, ie. 200 ug/ml for ZnT vs 1500 ug/ml for gfp.
I did question whether the source of my problem might be the kanamycin stock. I purchased more from Sigma (#K1377). I have increased the concentration of kanamycin in my agar plates and in the SOC media to 60 ug/ml. In the attached figure, I show different colony growth patterns of E. coli +/- kanamycin and +/- the kanamycin resistance plasmid. Does this suggest that the E. coli are kanamycin resistant at some level? Is there contamination of my E. coli stock?
I have expanded the E. coli from older frozen stock and made them competent using MgCl2 and CaCl2. These cells have worked previously, and still work as expected with ampicillin resistant plasmids
https://www.researchgate.net/post/Reason_for_spurious_colonies_on_kanamycin_plates2
Do they recommend only 10µg/ml for your plasmid? That seems quite low and could be the reason that you have such growth on your plate without plasmid. Check with the plasmid source what concentration to use, most will support growth up to 35-50µg/ml Kan. You could just try 3 or 4 concentrations to plate your cells and control (no plasmid) cells-use only the concentrations that give you no colonies from the mock transformation but still give you colonies with plasmid.
Looking at your plates at 60µg/ml Kan the 'no plasmid' grwoth suggests that there is something wrong with your Kan stock or you are adding too large a volume of cells and media and not allowing them to dry before inverting them. If plates are too wet growth is supported in the media on the plate surface until the cells contact the Kan in the plate itself.
There is also the possibility that you somehow have leaky expression of a toxic gene but I think that is unlikely in Dh5 cells.
Remember that not all plasmids will give you the same yield of miniprep, e.g. here the pCDNA3.1 has a pUC ori so is high copy number, if your Kan vector is pET based it will probably be low copy number.
Do they recommend only 10µg/ml for your plasmid? That seems quite low and could be the reason that you have such growth on your plate without plasmid. Check with the plasmid source what concentration to use, most will support growth up to 35-50µg/ml Kan. You could just try 3 or 4 concentrations to plate your cells and control (no plasmid) cells-use only the concentrations that give you no colonies from the mock transformation but still give you colonies with plasmid.
Looking at your plates at 60µg/ml Kan the 'no plasmid' grwoth suggests that there is something wrong with your Kan stock or you are adding too large a volume of cells and media and not allowing them to dry before inverting them. If plates are too wet growth is supported in the media on the plate surface until the cells contact the Kan in the plate itself.
There is also the possibility that you somehow have leaky expression of a toxic gene but I think that is unlikely in Dh5 cells.
Remember that not all plasmids will give you the same yield of miniprep, e.g. here the pCDNA3.1 has a pUC ori so is high copy number, if your Kan vector is pET based it will probably be low copy number.
Hi Craig! Probably a silly question, but are you waiting for LB to cool before adding the antibiotic?
It seems that the only reason you see colonies on a Km+ LB is some kind of contamination. Can you try with some other cells?
Beside making sure the LB reached ~50C before adding the antibiotic, and adding more kanamycin (30ug/ml sound right) I would suggest trying empty plasmid. Membrane proteins tend to be toxic to E. coli and leakiness, even without the T7-polymerase could be an issue. If this is indeed the case, try to work at 30C after transformation or even at 25C and let the bacteria grow a few hours more.
Yoram
Thanks for the suggestions!
I am confident that the agar is not too hot - I've often waited so long that the agar set in the beaker before I can finish pouring plates.
After reading on Researchgate, I have increased the concentration of kanamycin in my media/plates. I don't know why my reference says only 10 ug/ml. I think I copied the page from Molecular Cloning or some other generic reference text.
I had considered that membrane proteins might be toxic. I have successfully amplified this plasmid previously, however.
I will ensure that the plates dry in future. Although I have been leaving them for >1 hours after pouring, I agree that this may be a problem.
I've found some frozen E. coli JM109. I will see what happens when I expand them.
Regarding "I will ensure that the plates dry in future. Although I have been leaving them for >1 hours after pouring, I agree that this may be a problem."
From my experience 1h after pouring will suggest wet plates (although you will see it only after incubation). We leave them o.n. at RT and then ~1h in 37C to dry, even better ~20 min open at 50C if you have such an incubator.
Yoram
Kanamycin is usually a very clean selection and 10 - 20 ug/ml should be enough.
Are you sure your DH5alpha culture is pure? I'd have a look at those colonies under the microscope and make sure that they're rods and have the characteristic aimless-looking motility of E. coli.
Just this week I've run into the same problem as a consequence of using somebody else's mislabelled DH5alpha stock. Fortunately I encountered this once before, about ten years ago (roughly when my profile picture was taken).
There is a good chance that your strain is Max Efficiency DH5alphaF'IQ from ThermoFisher, which is kanamycin resistant due to a Tn5 transposon insertion. I have attached the product information.
This strain was produced by Invitrogen and is now owned by ThermoFisher. It can be hard to find information about it because in most cases it has been superceded by a tetracycline-resistant equivalent (e.g. NEB's equivalent strain).
I am not aware of any other kanamycin resistant DH5alpha cloning strains.
Thanks James. That does seem likely - we've given Invitrogen/LifeTech/Thermofisher a lot of our grant money in the last 10+ years, so it could be a poorly-labeled tube from a previous postdoc.
The JM109s solved the problem. For now....
Hi Craig,
I am kind of having the same problems too. I am seeing colonies in my untransformed plates with 10ug/ml of Kanamycin. I had expanded Stabl4 cells from previously prepared stock, and made them chemically competent. I used these cells to transform, but my untransformed plates are showing colonies. Is contamination to be the problem here?
Hello Indumati,
If there is something growing in kanamycin plates with untransformed bacteria, that suggests contamination with something that is kanamycin resistant. Alternately, your kanamycin is ineffective. See Yoram's and Nick's posts, above.
unspecific ligation of the vector??? what are the resction enzymes you use to cut the stich extremities?
I agree with Craig. Kanamycin resistance is a normally a very clean selection. You shouldn't see any growth of untransformed DH5-alpha at all.
I suggest you take a look at it under the microscope (40 or 100x) and check that you have rod-shaped bacteria. Yeast grows very well in E. coli media!
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