Hi All,
I have a pure plasmid with my gene of interest. The plasmid contains the a strep resistance gene for selection.
I am using two different hosts:
1) Rosetta-gami™ 2(DE3)pLysS (host has Cam resistance)
2) SHuffle® T7 Express Competent E. coli (no host resistance)
Every time I do a transformation, I get a biofilm of E. coli rather than single colonies.
For the SHuffle stain, I diluted 1:10,000 before plating on agar and still got a biofilm.
My question is:
1) Why is this happening?
2) Does it matter that it is happening since the plasmid is pure? i.e., can i just go ahead and inoculate?
Thanks for your help!
I do not understand. Your E. coli develop biofilms on agar plate without you setting the conditions for biofilm development? I hope you are not confusing microbial lawn development from heavy cultures with biofilm formation. Your photographs on plate do not show biofilm formation. I am seeing a very unprofessional attempt at streaking on the plates. Maybe I am missing something.
Maurice Ekpenyong
Your E. coli develop biofilms on agar plate without you setting the conditions for biofilm development?
- Correct.
I hope you are not confusing microbial lawn development from heavy cultures with biofilm formation.
- I am not getting single colonies. The question is why.
I think it depends on how you streaked ur plate or the cell/organism just clone that means it forms into colony but is still a single cell
The approach of streaking may be hampering it.
Just a suggestion: Use spreading method using a L-spreader else if you are very sure about the transformation rate to be high, then making a single line streak is can also do your work.
try to dilute with fresh media to 1000 fold and plate it (spread).
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