Which is the best method for transfection?
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It may be challenging to use large BAC DNA for transfection, although I have been interested in this idea as well. The main issue is that you have to get enough quantity of clean DNA without any contaminants such as endotoxins, but the BAC vectors are designed for low copy number replication. The simple way is to use a commercial BAC DNA kit such as Clontech BAC100 or Clontech MAXI XTRA. Both work great in my hands and I can get 100-300 ng/uL of BAC DNA on a regular basis. Don't over dry the pellet or you will not get a good yield. Always store at 4*C and don't freeze intact circular BAC DNA.
The other more complicated way is to clone the BAC fragment of interest into a new vector designed for higher copy number or inducible high copy replication. There is a system called CopyControl from Epicentre designed for this purpose. You should be able to get very high DNA yields with this method to maximize your chance of a good transfection.
Another thought I had to possibly increase transfection efficiency is to use polyamines to condense the BAC DNA prior to transfection. This is already known to work well for increasing the success of BAC DNA microinjection by avoiding shearing of the uncondensed DNA due to the adoption of a more tight configuration. Seems logical it might help with transfection or electroporation. With regards to the last point, it typical transfection methods fail you should move to electroporation if the cells can tolerate it. Maybe you don't need high efficiency or high survival but just want to see a few cells that get the BAC DNA taken up.
The other more complicated way is to clone the BAC fragment of interest into a new vector designed for higher copy number or inducible high copy replication. There is a system called CopyControl from Epicentre designed for this purpose. You should be able to get very high DNA yields with this method to maximize your chance of a good transfection.
Another thought I had to possibly increase transfection efficiency is to use polyamines to condense the BAC DNA prior to transfection. This is already known to work well for increasing the success of BAC DNA microinjection by avoiding shearing of the uncondensed DNA due to the adoption of a more tight configuration. Seems logical it might help with transfection or electroporation. With regards to the last point, it typical transfection methods fail you should move to electroporation if the cells can tolerate it. Maybe you don't need high efficiency or high survival but just want to see a few cells that get the BAC DNA taken up.
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