After ligation, we got 50 colonies on L.B + Ampicillin plate, we select all colonies for gene expression study at flask level but we did not get expression at all. Recombinants are resisistant to antibiotic therefore colonies grown but what is the actual problem, how we can troubleshoot the problems of these satellite colonies?
When a culture has ampicillin resistance, it will secrete beta-lactamase in the surrounding medium. The secreted enzyme then degrades ampicillin present in that zone. In this zone, growth of dormant bacteria begins, which form smaller colonies compared to the colonies containing resistance marker. These colonies look like satellites around the true transformant.
I would suggest you to use same concentration of carbenicillin, instead of ampicillin, as ampicillin is more prone to degradation by heat and low pH of the medium. Whereas carbenicillin is much more stable. Hope this is helpful.
Good luck... :)
Do you follow gene espression of a particular gene? Perhaps it's a problem of transcription. Are you sure your gene is in frame?
If your clones are resistant to antibiotic you can't call your clones "satellite colonies". Satellite colonies are sensible to ampicillin (or antibiotic used) and appear when the concentration of antibiotic is down.
I hope you are solution
Hi,
did you induce correctly, is you plasmid ok (see @sabine), also 50 is not too much for a re-transformation, check transformation efficiency. Also which strain are you in? Try different ones if you run into an expression problem.
How do you check expression? Blot? Sometimes we see very, very low expression, but get enough from our purifications.
Hope that helps a bit.
cheers Sven
Hi,
Shridar is right about the most likely origin of the satellite colonies. You can try plating fewer cells or reducing the incubation time to reduce the number of satellite colonies you see. You can also increase the concentration of ampicillin in your plate medium (e.g. 150-200 ug/ml instead of 50-100 ug/ml) or even try replica plating. The satellite colonies should not form new colonies on the replica.
Reduce the incubation time...That is sufficient.As soon as you find single tranparent colonies ,stop the incubation.Transfer your plates to 4 degrees.
The satellite colonies should be easily distingushed from the real transformants from the position and incubation time and replating. If a tranformation experiment obtained small number colonies only, for example, less than 50 colonies and these colonies showed resistance to the antibiotics but thete is no target gene expression, you also should think about the possibility of contamination. to confirm this, you can do a plasmid isoaltion and digetion to see if the plamis is still correct, or do a sequencing to see if any mutation happened during cloning or you gene is still IN FRAME.
For the bacterial colonies,14 to 15 hours in enough for colonies to come up on the plate. As soon as you find tiny colonies you can patch them on a fresh ampicillin plate and keep for 6 hours to get good growth of your colony.
Try dephosphorylating the vector and also check if you get some DNA in these colonies if nothing (increase you antibiotic) and i suggest be sure about the product rather than wasting on check the expression of these colonies. Try also playing with the Vector Insert ratio for cloning. Good luck.
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