12 replies to this topic

[lotus]

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?

[phage434]

Most digestions are near complete in 1/2 hour.  Why would you want to wait overnight?

[Vini]

lotus, on Nov 26 2009, 05:11 AM, said:

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?

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....

[Prep!]

lotus, on Nov 26 2009, 05:11 AM, said:

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?

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!!!!  

[Vini]

Pradeep Iyer, on Nov 26 2009, 03:49 PM, said:

lotus, on Nov 26 2009, 05:11 AM, said:

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?

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...

[Prep!]

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!!!

[Vini]

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  

[Prep!]

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!!!!  

[Vini]

Yeah Lotus.....hope we haven't further increased your confusion.....all the best....
I thought PI, that we were gonna start another thread here....

[Prep!]

    

hey now lets move out or else lotus s gonna get mad at us!!!!
sorry lotus and all the best with your digest!!  

[lotus]

Thank you everyone!

[toprak]

I do a lot of overnight digestions especially if I am digesting 15 ug of plasmid SNa for subcloning so that I can ensure 100% digestion, but I always use a total volume of 50ul for overnight digestions. and I keep the enzyme volume under 10%. I never had any star activity problem and I did do a lot of subcloninig.

[OA17]

I also know some people who leave the digestions O/N at room temperature instead of 37 ºC, and they say it works fine.

The only time I left a digestion o/n, was with Eco91I, and I did it at 37 without having problems with star activity or anything.