This question feels too stupid to ask but ... I have a plasmid that is available commercially but I would like to add a selection marker. I've constucted the plasmid but I am in a time crunch and would like to be able to order it online and get it back suspended and ready to transfect into my cells. Any recommendations?
Armina, you are not reading your plasmid properly. You already have the selection marker. It is neomycin/kanamycin resistance in your plasmid. Simply order https://www.thermofisher.com/order/catalog/product/10131027 (G418 Sulfate) First determine the kill curve in your host non-transfected cells and add the lowest concentration that kills 95%+ of untransfected cells after transfection. The typical concentration is 200–500 µg/mL depending on the cell type, after selection, you can usually add 30-50 µg/mL for maintenance of the culture (so non-transfected cells surviving initial selection would not proliferate).
There is also an AAV2 packaging sequence which means you can produce an adenoviral vector with it. Probably you know it already.
If you want to add the fluorescent tag you can do it with restriction enzymes, I have mapped the plasmid with the restriction sites. There is good suitable sites for insertion: MluI 139 and MFEI 452. When you do restrictive digestion you will see your main plasmid (large band) and the cutout of 313 bp (small band) on the gel. After you cut the large fragment (you can leave the cutout on the gel you don't need it) and extract DNA from the gel plasmid mix with ordered oligo sequence of GFP (see attached). You can also cut GFP from some other plasmid in your lab using restriction enzymes rather than ordering oligos. You can read more about the procedure here: https://www.addgene.org/protocols/subcloning/ Note that this GFP at this position will be simply produced into cytoplasm making cell fluorescent, but not marking anything in particular. Helps if you want to use flow sorting instead of selection with G418.
Also, install the SnapGene at least a free version to have a general idea of what is encoded on your plasmid and what to do with it. http://snapgene.com/
Hope I was able to help.
Thank you, I did modify the plasmid to have the selection marker but I don’t have this product in my hands
- Badges
- Science method