Hi,
I have a plasmid and designed site-directed mutagenesis primers, but my professor wants from me check plasmid with GFP after transformation? I donot know how to do that. Because our plasmid has not GFP sequence. In additon, I donot think so that I can add GFP sequence with mutagenesis primers. Can you give any advice or article or protocol for that?
Hello Merve Gündoğdu , is it possible to share the plasmid backbone image? Maybe someone may see something.
GFP is big, it cannot be PCR added on using a primer.
You may add GFP either as fusion protein or use a bicistronic vector. Alternatively add an expression plasmid with GFP (or another fluorescent protein) to your transfection, so you have an overall efficiency control.
You could do a simultaneous mutagenesis and insertion of GFP as a fusion partner as a three-part Gibson assembly reaction (e.g. ins 1= mut.F to POI-GFP. junction, ins 2= POI-GFP. junction to GFP. backbone.junction, ins 3= GFP. backbone.junction to mut.R), but given the fact that you only have your mutagenesis primers, I somehow doubt your mentor has this in mind. Perhaps the best course of action would be to ask the mentor what he/she meant by "checking the plasmid with GFP" :)
Good luck!
Go back and talk with your professor. I'm guessing that there is a misunderstanding about what plasmid you are using and how/what they want you to check.
My best guess is that they want you to use PCR to check for the presence of your insert, but it's best to ask.
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