First, I performed co-transformation using an electroporator device and then picked 15 colonies and cultured them overnight in LB media containing the antibiotic that the recombinant plasmid is resistant to.
I then harvested some of the cultured samples to do a DNA mini prep, and used DNA agarose gel electrophoresis to identify the bands.
However, I had a problem.
Out of the 15 samples that were incubated by colony picking, only 2 showed bands, and the rest of the samples did not show any bands.
Thinking it might be a DNA concentration issue, I raised the concentration and load them again, but the same problem occurred.
The samples were all run under the same conditions, but I can’t see any bands in 13 samples.
Some possible issues I can think of are that the LB agar plates that were treated with antibiotics were plates that had been refrigerated for 6 months, and I question whether this could have that much of an effect. (I used 50ug/ml kanamycin LB agar plates.)
The other prediction issue is that I picked satellite colonies, which is also a questionable prediction because I’ve never seen bands not appearing at such a high rate before.
Additionally, when I measured the DNA concentration through the nanodrop, I didn't see any issues with purity or DNA concentration.
Why does it appear as no band when electrophoresed on a DNA agarose gel?
Your agar plates are too old & the kanamycin degraded. Once poured, you have about 1 month in a refrigerator.
This is why you should take the time to read protocols in detail before you start an experiment. You need to know if your antibiotic is light-sensitive, temperature-sensitive, gives satellite colonies, etc. These are essential details.
As my Ph.D advisor said "you can't rush quality"
And pour your own plates
Good luck!
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