I've cloned a 1kb fragment into pet28a vector. Along with my positive colonies, I find a lot of white spurious colonies growing on the plates. These colonies are much smaller than my dh5a colonies and do not carry any plasmid. I've changed my kanamycin stock multiple times and also my competent cells. From what I understand, kanamycin is not prone to satellite colonies, so what could be the reason for this contamination and how do I get rid of it?
Whatever these colonies are, from your description it looks like they are definitely not DH5a, or even E. coli. E. coli are rapidly mobile, rod-shaped, gram-negative cells. Staphylo- or Streptococci are immobile, round cells and will stain gram-positive - if you really want to take it that far ;). You may also pick one colony, grow it in liquid culture, and look whether or not they are mobile. Other than that, either live with it, or eliminate the source.
Your observation (that the number of spurious colonies correlates with the number of positive clones on your plates), makes simple contamination of agar/media an unlikely possibility, but... who knows. These guys must come from somewhere.
Some suggestions:
Pick one white colony and streak on a fresh Kan plate. Will they grow without E. coli "feeding" them (with whatever)?
Plate a dilution of mock-transformed (no plasmid) DH5a competent cells on Kan plates, and on plates without antibiotic. White colonies?
Use competent cells of a different strain, e.g. DH10b (streptomycin-resistant), or NEB Turbo (tetracyclin-resistant). Plate your transformation on Kan/Strep or Kan/Tet plates, resp. White colonies?
DH5a has gyrA96 genotype = resistant to nalidixic acid, and probably other gyrase inhibitors. If your batch of competent cells is actually the source (against all odds...): To clean it up, make new competent cells using this selection marker.
Good luck!
It is either a contamination inside the incubator, or the enrichment or growth medium you use is contaminated.
You can still get satellite colonies on Kan. What concentration of Kan are you using? How long are you incubating your plates for?
The growth of colonies that do not contain a resistance marker suggest a problem with your selective conditions. If you are sure that the kanamycin stock is good then it is possible that it is becoming inactivated during the making of the plates by adding the antibiotic while the media is too hot. Have you tried plating out a strain that you are sure is kanamycin sensitive onto some of your plates to see if you observe growth if there are no resistant colonies on the plate?
Make sure the Kan is added at 50C, and try a higher concentration, or titration of it. Sometimes you can get satellite colonies with antibiotics other than Amp.
What is the concentration of Kan? and what type of media are you using? I agree with Adam Mott, try to run a Kan sensitive strain on the same batch of media to make sure that your conditions are optimal.
1. If you pour your own plates, you could very easily have a contaminate due to improper autoclaving (or malfunction in the autoclave), or the plates were not allowed to dry/cool completely in the sterile hood before storing, thus allowing condensation on the lid and particulate contaminates.
2. Your concentration of Kanamycin is too low. Try a range between 33-66ug/ml.
3. Did you use any controls? Specifically, plate 1 = DH5A only (-ctrl), plate 2 = transformant of interest, plate 3 = kanamycin resistant strain (+ctrl)
Make sure you do an overnight growth exactly. This means 16-18 hours. Not more than 18 though!! I usually do my transformations in the afternoon so that I plate my transformation mix around 5-6 pm. Next day collect them at 9-10am.
to Alexis Varin: i ve changed incubators. others in the lab use the same SOC medium and incubator without any contamination
to Stephen Doyle, Hosni Hassan: im using 50ug/ml kanamycin in LB agar. incubating the plates for 12-14 hrs
to Koen Venken, Adam Mott, Joshua Geltz : I prepare my own plates however im sure ive not added the antibiotic while the media was too hot. The autoclave is fine cuz others in the lab are using the same autoclave and not getting contamination on their plates. the plates were allowed to dry for 30 mins in laminar hood before sealing them(inside the hood).
to Joshua Geltz ,Adam Mott ,Hosni Hassan: thanks for the suggestion. i will plate plain old DH5a on the Kanplates to see if anything interesting happens. im also going to increase the conc of Kan and monitor after how many hours these "small" white colonies come up. im planning to not let the plates sit for more than 7-8 hours, cuz i suspect the contamination comes only after my positive colonies have grown.
to Stephen Doyle: i'm not familiar with satellite colonies on Kan plates. can you please elaborate why that happens.
To Priyamvada Jain, I would expect there will nothing on the Kanplante when you plate DH5a. Curious why this happen....
What is the location of those small white colonies on your Kan plates? So, we can diagnose this situation better.
I think It's a contamination. Probably It's Staphylococcus epidermidis or similar. It's a nonpathogenic commensal and it lives on the skin. After overnight incubation, Staphylococcus has very small colonies and it often carries resistence genes of aminoglycoside. You can try to plate one of these small colonies on mannitol agar salt or add 75 g/L of NaCl in the same agar broth that you usually use for your trasformation.( I guess LB agar). If, after incubation at 37°C, you have a growth most probably It's a Staphylococcus.
@Seyda Acar - completely agree - it is not "overnight" at 12-14 h. All of my colonies would look small and "satelliite" SIZED at this time. I follow a similar timeframe. ~16h is good
@Priyamvada Jain - Kan is not prone to satellites as Amp is - by far - but it can still happen. The mechanism of generating is different that results in satellites between the two antibiotics (and I am not sure exactly how it happens with Kan), but I imagine it is the result of sub-optimal selection due to a number of factors raised above, ie concentration, quality, heat etc. Personally, I use 100ug/ml stocks, which help.
Satellite colonies typically describe the pattern of small colonies around an established colony (although they can become more diffuse over time) - i agree with @Ender Cerit that if you describe what they look like a bit more, you might get a more specific diagnosis.
Also, when you say you have changed the Kanamycin stock, does that mean you went back to the freezer and grabbed a new but previously made-up aliquot? Or did you go and get the Kanamycin powder and make up a NEW stock for yourself (providing the powder is also now very old)? It is a possibility that someone has made up the stock incorrectly (or they are very old)??
to Ender Cerit , Stephen Doyle: these colonies are much smaller than my positive clones, and white in colour, they are present in the vicinity of my positive colonies. usually if my transformation efficiency is good and i get a lot of positive colonies, the contaminants are spread over the entire plate. if i get a few solitary positive clones here and there, then such areas are devoid of any contamination. dats why i felt they resemble satellite colonies.
I made a new Kan stock right from the powder, with filter sterilized and autoclaved miliQ water inside the laminar hood.
Whatever these colonies are, from your description it looks like they are definitely not DH5a, or even E. coli. E. coli are rapidly mobile, rod-shaped, gram-negative cells. Staphylo- or Streptococci are immobile, round cells and will stain gram-positive - if you really want to take it that far ;). You may also pick one colony, grow it in liquid culture, and look whether or not they are mobile. Other than that, either live with it, or eliminate the source.
Your observation (that the number of spurious colonies correlates with the number of positive clones on your plates), makes simple contamination of agar/media an unlikely possibility, but... who knows. These guys must come from somewhere.
Some suggestions:
Pick one white colony and streak on a fresh Kan plate. Will they grow without E. coli "feeding" them (with whatever)?
Plate a dilution of mock-transformed (no plasmid) DH5a competent cells on Kan plates, and on plates without antibiotic. White colonies?
Use competent cells of a different strain, e.g. DH10b (streptomycin-resistant), or NEB Turbo (tetracyclin-resistant). Plate your transformation on Kan/Strep or Kan/Tet plates, resp. White colonies?
DH5a has gyrA96 genotype = resistant to nalidixic acid, and probably other gyrase inhibitors. If your batch of competent cells is actually the source (against all odds...): To clean it up, make new competent cells using this selection marker.
Good luck!
Had kind of the same problem once. However I didn't dwell on it. My kanamycin plates were rather old. I simply made a new kanamycin stock solution, picked one of the large colonies and made a starting culture with 5 ml LB-medium containing 33 µg/ml kanamycin following a larger over night batch culture (with antibiotics). Checked the plasmid DNA I isolated on a gel and it was just fine.
Koen Venken is correct and often the Kan stock is fine but people can have different ideas of how hot the agar is. You could be adding the Kan to the agar when it was too hot and the Kan gets broken down. This kept happening to one of my students until I told him to make sure to pour the agar into a room temperature sterile 50 ml conical and letting it cool to 50 C or below before adding the Kan. After this change his satellite colonies stopped growing. Good luck!
I agree with alexandra lepier, try to "clean" your DH5a stock... I had the same problem as you and after doing my own competent cells using my own chemicals, everything went better :-)
Please forget about the small colonies, just to screen the ''right'' colonies. Don't west your time on it. When the transformation is not well done, the satellite colonies sometime happen whatever antibiotics you used.
Alexandra Lepier has covered pretty much every possible problem in your case. You should try those. Remember, most of the time, the resistance genes in transformants enable the bacteria to degrade the antibiotics around the colony. That is why the bacteria without the resistance genes can grow on that area. If that description fits your case, your small spurious colonies are most probably satellites.
You can pick few cells from the colonies on the plate, put on a slide and stain with crystal violet (simple stain).
You can see the shape of colonies and observe that some are not E. coli , ( cocci , or long rods or oval shaped cells).
We have noticed over the past few years that kanamycin is no longer as tight a drug resistance marker as it used to be. Make sure that you are not adding the drug to the agar when it is too hot. The drug is not happy at high temperatures. Background colonies are also somewhat dependent on the host strain you are using. As crazy as this sounds, I think that they manufacturing process for the drug has changed and it is not as potent as it used to be. Real Km transformants can be distinguished from false positives by seeing if they grow flat on the surface of the agar. If they colonies are flat, they are not real transformants.
Sidney Kushner
I agree with those who have said you can get satellite colonies with Kanamycin. The Agar really does need to be cooled down before addition of Kanamycin preferably in a waterbath so that it is evenly cooled. If you pick a couple of these colonies you will probably find that they do not grow in your liquid media with Kanamycin present as it is easy to add antibiotic to cold liquid so no degredation of kanamycin occurs. Also your Kanamycin powder can go off too, therefore new stock will not be fully effective.
As far as I remember E. coli B strains are naturally resistant to kanamycin to a certain extent, as shown by appearance of tiny colonies upon prolonged incubation on kanamycin plates (several days). This is not the case with K strains. How long did you incubate your plates? You can find useful information about the multitude of E. coli genotypes on the openwetware website http://openwetware.org/wiki/E._coli_genotypes#DH5.CE.B1
As already suggested check out the kanamycin concnetrations etc. but also leave your plates in the face up position until there is no obvious plating liquid left (if you tilt the plates you do not want to see drops running across the surface). Sometimes what look like satellites are just cells that have begun to grow in the excess media used for plating before they can be selected by the Kan in the plate .
Make sure that the Km is added at ~ 50C, and use a fresh stock of Km. Also, take in consideration that the commercial powdered product (i.e., Kanamycin Sulfate) contains only 67.7% active Kanamycin. That means for 50ug/ml you need to add 74 ug/ml of the Km Sulfate powder.
Hello Priyamvada ...
In case of PET 28a, sometimes using large amount of culture for plasmid isolation works to show the plasmid on gel.
Best
Hi Priyamvada
I am having a same problem that you had faced. Did you find a way to solve it?
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