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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.

-mbsea-

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.

-Papaver-

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

-mbsea-

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...

-Papaver-