Hello, this is colony PCR i did by using gene forward and reverse primers. My question is why some colonies are small and some are big? What kind of colonies i use for plasmid extraction and sequencing? My gene product is 972bp and it is in pEasy blunt T vector (3596 bp). Thanks
In my case i used kanamycin, over night at 37 degree, and i did transformation quickly after ligation means not keep plates for long time in incubator. So what is real problem in my case? Can i choose big colony and sequence it?
Unless they represent contaminant bacteria or satellite colonies, do not sweat about colony size when picking clones for minipreps/sequencing. After all, you will grow those liquid cultures to saturation anyway. The yield/sequence quality will be nearly identical.
I'm guessing there may all sorts of different causes behind this variation. In some cases you observe two clearly different populations (one small, or smooth, or whatever and the other large, rough, etc), and when you pick an individual colony and re-plate it, you again get the two populations; this is clearly some sort of phase variation involving a gene that ultimately influences colony phenotype. Other times you simply observe heterogeneity in colony size, i.e. gradations from the smallest to the biggest that tend to even out as time passes; in this case I would think that the differences arise from founder cells in different stages of the cell cycle or simply in different physiological states (a dividing cell, for instance, would have a headstart over another where DNA replication is arrested due to some temporary stress).
Often, replating individual colonies of different sizes will tell you whether there is a contamination or not.
Hope it helps!
Hey why I am getting bigger colonies on ampicillin plate while growing pRSETA Vector in dh5alpha
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