I am cutting a certain plasmid with Pst1-HF and Cutsmart buffer for cloning. The digest seems to work efficiently as verified by Gel Purification. Following this I combine the cut plasmid along with NEBuilder and the two geneblocks I am using (replacing the gene blocks with nano pure H2O for my negative control), incubating for 15 minutes at 50°C. From then I follow a transformation protocol using NEB5-Alpha cells and plating on LB Kanamycin. I have attempted this twice already, but am still having growth on my negative control even though there are no gene blocks present. Does anyone have any idea why this may be occurring?
Hi there,
The PstI digestion of the initial plasmid is not total. Are you sure to separate properly linearized and circular forms of the plasmid?
Dominique: I ran my cut vs. uncut plasmid parallel with a 100bp - 10kb Versa Ladder. From there I selected the linear fragment.
Hi again, what amount of plasmid do you engage and how long do you digest it? What is actually distance on gel between the 2 species? As you wrote you "selected the linear fragment" it means you have several bands in your digest which is therefore partial.
Even if you get clones on your control plate, it doesn't really matter provided you get significantly more clones with your cloning experiment (for instance, if you 10 clones on your control and 100 in the cloning experiment, it means that 90% of the clones should be positive).
Dominique: thank you for the response. I did get at least 2-4x more clones on my -C than actual experimental plate, which is what concerns me. I used 4.82μL of my uncut plasmid and digested the protein for 2 hours at 37 °C.
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