I have isolated the plasmid in pure form by using commercial kit. Then I performed double digestion of the same plasmid with Not I and Eco RI enzymes. After gel electrophoresis I got band in control (uncut plasmid) lane but no band in treated lanes. I have repeated the experiment many times by altering the parameters like enzyme conc., incubation time and pretreatments of plasmid, but got nothing.
You are right Mr. Arjan. After digestion, the plasmid disappears. I did have loaded the uncut plasmid in the next well, but unfortunately I didn't get any band to compare with uncut plasmid. Actually my insert is having the ligation sites for Eco RI and Not I, that's why I am treating the pET22b+ with Eco RI and Not I. I also tried the sequential digestion method, but that too didn't work. To cross check enzyme quality, I used same enzymes in other experiments and got the positive results.
It sounds like your DNA has EndA nuclease contamination, and is degrading completely in the digestion step (or possibly during prior storage). This is a common issue for pET plasmids isolated from BL21 cells; most BL21 strains are EndA+. Try retransforming into JM109 or DH5a as the host strain (BL21 is fine for protein expression, but a poor choice for cloning).
make a fresh DNA prep of your plasmid and be sure you are away from DNAse.
Thank you Mr. Daniel. As per your suggestion, I have retransformed the plasmid in DH5a successfully. No will go for isolation and restriction digestion. Hoping positive result... Thanks again.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression