I'm working with a 100 kb plasmid and have been trying to digest it using BamHI along with other available restriction sites. I've attempted digestion for conventional 3 hours at 37°C, as well as overnight, but the plasmid remains undigested in both cases. Has anyone encountered a similar issue or have suggestions on how to improve digestion efficiency?
Let's see if you can rule out any possible issues:
You sure the restriction enzymes have degraded or expired?
Double-checked your math to make sure you are using the correct amount of each reagent?
What volume are you setting up?
How much DNA are you using? You can have too much or too little.
@katie A S Burnette thankyou for your response.
Restriction enzymes are fresh and in working condition I have used control to check them.
Total reaction volume is 50 ul in which I am adding 2-5 ug and restriction enzymes are 1.5ul. Please suggest what else I can change or do.
100kb plasmids are very difficult to isolate and purify in significant amounts, especially since they are low copy number. I really wonder whether what you are looking at is really your plasmid?
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