The answer may be as obvious as there must be something wrong with the plates, but I am still perplexed. I have a plasmid system which has kanamycin resistance on the plasmid vector while expressing chloramphenicol resistance on the antisense strand of RNA produced. I did not expect growth on my plates with CAM or CAM and KAN. However, I did expect growth on LB and on KAN. To my surprise however, the first time I had tried growing my DH5alpha cells from NEB after Gibson cloning of large vector backbones/inserts (around 7000-8000 bp in the final product) nothing had survived. However, after leaving my transformed cells in SOC at four degrees for two days I tried plating again on different plates.
None, however, grew on the LB + Agar plates while I have colonies on my all my new KAN plates. The only noticeable difference between all the plates that I had used and these KAN ones is that they are a much darker yellow, making it seem as though they have a much higher LB content than the other plates. Might this at all be the reason why I have growth on these plates but not the others? Or could something else be the culprit?
Any advice would be appreciated. Thank you!
This sounds very weird to me. You might be growing a contaminant. In my experience this kind of cloning works poorly in dh5 alpha even more so when you have large inserts. Remake the plates and transform you GA mix into neb-10-beta instead.
I would like to suggest you, check the KAN vial, it is outdated or it has contamination or expire etc..
DH5A cells (whether transformation was successful or not) should be able to grow on LB plates without antibiotics. Since you had no growth on LB plates (your first try) suggests your cells are not viable. Are you sure your cells are alive?
No growth on LB plates suggests you have no viable cells in your SOC medium. Colonies on Kanamycin plates are most likely contaminants. Check your stock kanamycin plates(if you have any stored) to make sure there is nothing growing on it. How many colonies do you have on Km plates? You can do a pcr to check if the colonies have taken up your plasmid.
There is no reason your strain would not grow on plain LB plates. But if the plates are truly an odd color as you mention, then it is highly likely someone made a mistake in the preparation of that batch of plates. Try some new plates first before getting too worried.
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