Recently, I do transformation in Kanamycin resistant plate and I observe a weird phenomenon. After incubation at 37oC for 16-20h of my ligation product, I only observed few colonies. However, the following day (36 hr incubation), I observe a bundle of colonies. It does not look like satellite colonies which I observed in the Ampicillin resistant plate if I incubate too long.
In the Kanamycin plate,the colonies look very normal. Is it satellite colonies or my target clone? I did not pick up the colonies and do sequence. Is there anyone can explain this phenomenon for me? Thanks
If the bacteria were spreaded thoroughly, these colonies are probably satellites. Depends on how close they are, really. I would pick the one in the centre (probably the biggest one) and search for your plasmid there.
I did the transformation again and pick the colonies after 16-20h incubation. Then I keep incubate to observe what happens and I observed many colonies in the next day. I did not pick these colonies. Is it satellite bacteria because they look very normal.
Why don't you test them??
A very easy way to test these colonies is just take a part of these colonies with a tip, resuspend it in water in a PCR tube, make a boil and prepare a PCR with primers of the plasmid to amplify the MCS, where you cloned your DNA fragment. This way you will be able to see if some colonies have a band of the correct size (your fragment + part of the plasmid's sequence amplified (depending on where your primers bind)).
I hope it helps
Thanks Anna! I just think this is satellite bacteria so I dont want to waste my time on it. I made a question here to check whether is there anyone has the same situation like this or not. They might have test them and have an answer :)
Ok, they can be satellite, but with kanamycin is rather infrequent (contrary to what happens with b-lactams, which are secrected extracellularly, and easly allow growth of satellite colonies).
Good luck!!
I do not think those are satellite colonies, as this does not happen with aminoglycoside-modifying resistance enzymes. If you really want to find out what they are, do as suggested above: pick a colony, reisolate on same medium and do a PCR:
- If the reisolated colony does not grow or grows very poorly (in more than 36h) on the kanamycin plates, those could be satellite colonies since they would be kanamycin sensitive
- If they grow on the kanamycin plate, you can PCR the marker to see if they carry the kanamycin -resistance marker. My guess is that they will not and that they are spontaneous knamycin-resistant mutants, which happen quite easily if you incubate your plates too long.
Good luck!
Hervé
Hello, dear Trang, this may be due to the low concentration or the loss of kanamycin during the preparation of agar plate. Make a culture medium with a higher concentration of kanamycin
@Trang. The bacteria might be highly resistance to the kanamycin which is less concentrated or the bacterial inoculum size is too turbit which means is not equivalent to mcfarland standard thereby making the bacteria to grow without any effect of the kanamycin. Since the antibiotic has no effect against the bacteria you will definitely see more colonies after 16-36hrs of incubation.
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