I used pvuII HF and pacI from NEB to digest my vector two three times and according to the sites I need to get two fragments but always I got the three four fragment don't know what is that. I also changed the enzyme lot but didn't got any specific thing. Please let me know how I can get this.
Thanks.
I have couple of questions,
which vector are you using and
incubation time of digestion
1) See the comment of Ajaz
2) decrease the units of enzyme per reaction (either by pipetting less, or by partial thermal inactivation) or incubation time
Star activity doesn't give extra fragments. It just cleaves extra bases from the site.
Double check the number of restriction site. Upload ur Vector sequence on NEB cutter and check the number of sites. You can also use other vector tools
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