I need to obtain high-quality pJETAsptRNA DNA plasmid to synthesize the oligos by using a T7 polymerase RNA synthesis kit. I am using Bgl2 digestion to access the plasmid DNA quality. But my DNA is not cutting within 1 hour. I use the rapid boiling method to miniprep plasmid DNA. Can you give me suggestions to improve the rapid boiling method to obtain high quality DNA
The issue is probably not the boiling mini-prep. What are you seeing when you run out cut vs uncut plasmid?
Are you using a known "rapid cutter" version of the enzyme? 1 hour might not be enough time.
How much plasmid DNA are you using for each reaction? I mean number of molecules/concentration not volume. It's possible to use too little to see on a gel OR to use too much plasmid for the digestion to go to completion in the time you picked.
Are you following the basic protocol for the enzyme? Check the buffer type & digest temperature. Make sure the total volume of the enzyme is less than 10% of the reaction volume & mix the reaction thoroughly. Otherwise, the enzyme in glycerol will sink to the bottom of the tube.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus