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Storing Ligated/Transformation Material before Transfection - (Aug/05/2015 )



I am trying to figure out the best way to store my ligation mixture before I perform a transfection.


I am hoping to clone my insert into pCDNA3.


My question is can I first ligate my insert into pCDNA3, transform into bacteria and make glycerol stocks with these colonies before I do the transfection?


Originally I was thinking of immediately transfecting after I performed the ligation. It seems like it would be easier though to prepare the DNA from the glycerol stock.


Thank You





-Jacob Black-

Make glycerol stocks so that you have a "never-ending" supply of bugs containing your plasmid. If you ligate and then use that for transfection, you will not only find that you probably don't have enough DNA to transfect, but also you won't have any left to transform and you will have to repeat the ligation step each time.


Also, transfection with a ligation mix would come with some problems - what about the DNA that doesn't ligate - how does that affect the transfection (linear fragments work better), and what about the ligation components such as ligase etc?