Hello ,
I transformed yeast cells with a plasmid containing URA marker and plated on SD-Glucose and Galactose plates.I obtained about 350 colonies on the glucose plates but only 100 on galactose plates.Is this usual to get fewer colonies on gal plates? and please suggest methods to increase the transformation efficiency on gal plates if there are any. Thanks
Hi there,
If you plated exactly the same sample from the same transformation mix on both media you should have get the same result provided cells may use either glucose or galactose indifferently as carbon source. But there are exceptions: for instance if the ura plasmid is encoding for a toxic protein under galactose promoter, you expect less viable clones on galactose...
Thank you, Dominique.
The plasmid does not encode any toxic product.I tried transforming multiple times on Gal plates and ended up getting only 1/3 of the colonies obtained on glucose plates. Is there any way to maximize the transformation efficiency on Gal plates..Please do share your suggestions
So you spread identical aliquots of one unique transformation mix on glucose and on galactose plates in parallel?
yes I had spread identical aliquots on glucose and galctose plates in parallel
Apart from the carbon source, plates were also strictly identical from a composition point of view? (I mean the same batch of agar media splitted into 2 and then supplemented with either glucose or galactose at the same final concentration (2%)). Does the strain present any defect in galactose use as a carbon source?
I used the normal BY4741 strain.
Isn't Galactose less preferred over glucose? and I find the cells growing very slow on galactose supplemented media.
Indeed yeast exhibit slow growth phenotype on galactose (roughly generation time is twice the one observed on minimal complete media with glucose) but viability should be exactly the same as the strain you use has no defect towards the use of galactose as carbon source...
Please check this out.They used glucose in addition to galactose to get more colonies so as to screen more library transformants. I too want to use a library under Gal promotor , therefore my aim is also to get as many colonies as possible on Gal plates
Is it a good idea to use glucose with galactose??
http://www.biotechniques.com/multimedia/archive/00010/01301st06_10260a.pdf
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