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Transformed Agro colonies growing very slowly

molecular Biologe Microbiology Genetics Biochemistry

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#1 awan

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Posted 22 February 2014 - 01:23 AM

Dear Experts

I Transformed Gateway plasmid pMDC83 with my 1.5 kb insert to Agrobacterium competent cells, GV3101. The agro colonies are growing after 7-8 days on LB agar plates containing kanamycin 50 microgram/mL and Rifampicin 25 microgram/mL. I have repeated the transformation 4 times. The colony PCR shows the presence of insert. should I proceed for the plant transformation?



#2 glez

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Posted 04 June 2014 - 08:31 PM

You need to tell us the temperature you are using in order for us to see if that is slow. Can I assume you are growing them at around 28C or 30C? If so, then 7-8 days is slow. Maybe your gene is slightly toxic. You might also have E.coli contamination. Rifampicin is light sensitive and degrades over time. Can you restreak the single colonies onto a new plate? If the restreaked cells grow within 1-2 days, then the colony was probably fine. I'd try plant transformations with that.


Edited by glez, 04 June 2014 - 08:32 PM.






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