Hi to all.
Hope all of you are doing well.
I am constructing Ez-tn5 (Kan2) transposon Library in E.coli to create mutants . I got ver very tiny colonies ( 15-20) on nagative plate also(water used) after 19 hrs of incubation. You can't see these colonies by first look. I had to focus below light to see these colonies (image attached). My tn5 transformation plates are ok and full of colonies( image of one tn5 plate is attached).
You can see the water vapours in negative plate. But there are no water vapours in tn5 plates. I am using Kanamycin as selection marker. When I restreaked 11 tiny colonies from negative control Plate on Kanamycin plate ), the next day I got colonies from one colony (colony#2) only,and from rest of 10 colonies I got no cololny ( images attached). Moreover, I incubated the nagative control Plate for additional 16 hrs ,and i noticed that ony the size of colony number #2 became enlarge but rest of the colonies remain same size.(image attached)
So, what do you think that either this contamination of colony#2 came due to water vapours or from somewhere else????
And please give me suggestion, either I should used this Tn5 transformation library to pool or not( I have total 18 big plates with Transposoms mutants😉)?????.As I got colonies from only one colony from negative control Plate when I restreaked it on selection plate.
Thanks in advance
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