I have a plasmid isolation that probably contains salt contamination. Due to the fact that , all my plasmid is digested because of Star activity at 50uL reaction overnight digestion.
I digest 3ug plasmid with 1 ul xhoI and MluI in fermentas R buffer.
When I digest short term I have no star activty. 10-12 h
may I reduce the star activity with 100uL total volume for same reaction or does that cause incomplate digestion? Do I have to digest shorter?
Best Regards
Hi there,
The following page from NEB should help:
https://www.neb.com/faqs/0001/01/01/what-is-star-activity-and-how-can-it-be-avoided
You said it yourself, when you shorten the incubation time the star activity disappears. Or are you getting partial digestion under that condition?
But in short term I got single cut vector that can self ligate itself with t4.
I have fix the problem with a new plasmid isolation. Probably a alcohol contamination during one of wash step, or salt contamination caused the problem.
Thanks for all answers.
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