double digestion with sac1, hindIII - (Oct/14/2011 )

Hi everybody, i have been struggling with my ligation of my gene into oet 21a vector. Now i changed my primers also enzymes. I want to use sac1 and hind III fermentas enzymes. I cloned my gene into pcr 8 topota vector. Did anybody use this combination and offer me a protocol that how long must i wait and how much enzyme must i use?

best regards.

Fermentas double digest: http://www.fermentas...ls/doubledigest
Select your enzymes, then fermentas show their recommendation and general properties of the enzymes.

I usually digest ~ 1 µg of template, 1xbuffer, 0.5 - 1 µl enzyme in a volume of 30 - 50 µl. Usually digestion at 37 °C for 2-3 h is enough.

thank you very much. I was wondering about the incubation time because i am not sure that my vector is digested well..
best regards...

If you are not sure how long would be ideal you can set up several digestions and stop the reaction after 1, 2, 3...hours. I made the experience that too long incubation and/or too much enzyme ends up in unspecific digestion of my plasmid. Too less can lead to uncomplete digestions which I prefere at the end as long as enough of my plasmid was digested properly. It's always learning by doing...