overnight restriction digestion- good or bad? - Should we modify amount of enzyme added for overnight digests? (Nov/25/2009 )
Hello,
Some people say overnight restriction digestions are bad because there will be evaporation of the buffer and so salt concentration will increase over time and also there is a possibility of star activity.
Has anyone faced such problems? Can we avoid star activity by adding lower amount of enzyme?
Most digestions are near complete in 1/2 hour. Why would you want to wait overnight?
lotus on Nov 26 2009, 05:11 AM said:
Some people say overnight restriction digestions are bad because there will be evaporation of the buffer and so salt concentration will increase over time and also there is a possibility of star activity.
Has anyone faced such problems? Can we avoid star activity by adding lower amount of enzyme?
Hey, o/n digestions can be bad depending on the enzyme u use...like NEB BamHI etc. do show star activity...I hv put up o/n digestions wid enzymes like NotI etc and they turn out to be fine........but o/n digestions are really not required unless u just wanna decamp from the lab....
lotus on Nov 26 2009, 05:11 AM said:
Some people say overnight restriction digestions are bad because there will be evaporation of the buffer and so salt concentration will increase over time and also there is a possibility of star activity.
Has anyone faced such problems? Can we avoid star activity by adding lower amount of enzyme?
well i think it has been well explained tat it can happen in a few hours max... so no need for a overnight incubation.. i am anyways not a genomics guy.. have done just a couple of restriction digests..but still if u wanna keep it overnight without evaporation best bet would be to keep it in a place were the surrounding temperature is more than the incubation temperature!!!!
Pradeep Iyer on Nov 26 2009, 03:49 PM said:
lotus on Nov 26 2009, 05:11 AM said:
Some people say overnight restriction digestions are bad because there will be evaporation of the buffer and so salt concentration will increase over time and also there is a possibility of star activity.
Has anyone faced such problems? Can we avoid star activity by adding lower amount of enzyme?
well i think it has been well explained tat it can happen in a few hours max... so no need for a overnight incubation.. i am anyways not a genomics guy.. have done just a couple of restriction digests..but still if u wanna keep it overnight without evaporation best bet would be to keep it in a place were the surrounding temperature is more than the incubation temperature!!!!
oh no! such temperature adjustments are not required.....eppendorf tubes have lids........just spin down the reaction and process...
hey DRN.... if you carefully notice the PCR machine.. the lid temperature is 104 degrees that is higher than the block temperature on which the tubes are placed... the lids are not fool proof.. overnight at 37 can lead to evaporation slowly from the lid too... unless it is wrapped around with parafilm!!!
so it is scientific!!!! and anyways tat was meant as a fall back if the overnight incubation is cumpolsary and the eveporation to be controlled!!!
Hey PI , I understand ur scientific concerns ..........but its my experience in genomics speaking, that its not such a big deal.....I also said that it if it has to be done due to whatever unavoidable reasons, it can b done........I hope I have made myself clear 
point taken DRN!!!! your expertise is noted!!! i also understnad the difference between practicality and theoritical experimentation!!! you seem to have taken the solution too much to heart!!! 
dont mind us lotus... but u got ur answers i guess!!!!
Yeah Lotus.....hope we haven't further increased your confusion.....all the best....
I thought PI, that we were gonna start another thread here....
hey now lets move out or else lotus s gonna get mad at us!!!!
sorry lotus and all the best with your digest!!