Hi everyone, I performed a miniprep to verify if a correct construct was made. The overall purpose of my project is to put RFP gene into puc19. I did Nanodrop to measure DNA concentration and the concentrations were pretty good (>20ng/microliter). Miniprep was done to isolate plasmid DNA and then I do a restrition digest with BamHI and EcoRI enzymes to verify that at least 1 colony has the desired insert (RFP). I used a master mix to make this digest reaction. This is the restriction digest gel showing 1 kb ladder in lane 1 and tubes #1-#6 in the other wells.
My question is that if this miniprep was successful and can we tell by the gel? What is the data telling us? Did a colony have the desired insert? Thanks so much for the help.
Plasmid isolation can be confirm by seeing the band pattern on gel like it shows two or more bands due to it circular and semi circular nature. After digestion it converts from circular to linear so we see single band. After ligation you can see the band shift on gel like the base pair of gene inserted into vector increases the size.
It is really hard to tell from that gel what you might have. First of all, the gel is not well stained nor has it been run long enough. I think the RFP gene is pretty small so it should be much lower in the gel than the plasmid band. I also can not clearly see the 1kb ladder. But your mini preps look pretty good. I would do your digests again and run the gel much longer so you get good separation (run the dye to near the bottom) and stain the gel better after the electrophoresis. Also how big should the insert be?
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