I used up GFP plasmid in our lab so I am trying to transform GFP plasmid into E. coli then extract GFP plasmid after culture.
I did transformation with this protocol
1. thawing E. coli in ice for 5 min.
2. co-incubation with DNA in ice for 30 min.
3. heat shock at 37°C for 45 sec.
4. add 1ml of SOC media
5. transfer to ice for 2 min
6. incubation at 37°C at 225rpm for 1 h.
7. take 100ul of E. coli and spread it on agar plate with antibiotic
8. overnight culture at 37°C
I saw pictures of colonies generating green fluorescence but my E. coli colonies don't show any fluorescence under the UV lights.
Is it normal? GFP plasmid I used is pBI221, promoter is 35S and antibiotic resistance is
ampicillin. I took 50ul of DH5a competent E. coli and used 28ng/ul of GFP, 100ug/ml ampicillin.
I'm going to do colony PCR and wish to know whether it's normal or not prior to that.
If there is a bacterial promoter before the GFP casette, GFP should be expressed and flourescence should be visible.
With a stronger promoter like t7/lac, these would be bright green by eye, even without any fluorescence. Your promoter is certainly weaker, but I do guess there should be some visibility under UV.
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