IGEM:Peking/2007/Switch: DNA digestion
endonuclease enzymatic reaction
There is great influence on enzymatic efficiency by enzymatic buffer. We should choose buffer according to the double digestion form provided by Takara and different activities for different endonuclease in various buffer.
Most endonuclease has the optimal activity temperature of 37℃. There is exceptions such as SmaI's best temperature is 30℃ while SfiI needs 50℃ to work efficiently.
Includs plasmid/PCR product, enzyme, buffer and water, sometimes with BSA and Trition X-100 in it. The system for enzymatic test should be no more than 20uL. The system for recruiting vectors and fragments should be around 100uL.
DNA concentration in digestion system
The vector concentration should be no more than 1ug, PCR fragment concentration should be no less than 1ug.
Some endonuclease will have a less specific substrate selection under certain conditions. It will cut different sequences from original recognitions. This is called star activity.
In order to reduce the star activity, glycero concentration should not be too high and DNA concentration should not be too low. In the storage of enzyme there is glycerol. So the maximal amount of enzyme added in a system should be 10%, especially for enzymes with star activities.
The influence of Methylation
Some endonuclease activity will be affected by methylation of DNA.
There is a methylation form we can refer to.
Terminal digestion of PCR product
The terminal digestion always has a low efficiency. Some additional base pairs are always added at the end of PCR product during primer design in order to improve the digestion efficiency.
There is a form about PCR terminal digestion.
After the digestion, endonuclease must be inactivated before applied to ligation system. Different enzymes have different inactivation condition.
There is a form about inactivation conditions of all usual endonuclease.
1 hour enables a test digestion. If digestion is used for cutting vectors and PCR fragments, in order to digest thoroughly, about 16 hours is a relatively good choice. However, some endonuclease used in the digestion system inactivates quickly. So we need add new enzymes at intervals during digestion.
There is a special form of different endonuclease about their remaining activity after different periods of time.
DO NOT carry the endonuclease out of fridge. If really need to do so, please use an ice box to protect the enzymes from inactivation due to high temperature.
Protection of cross-pollution between different enzymes
If endonuclease have already been added into buffer solution while somehow the digestion reaction is not ready to start right now, DO store the mixed solution under 4℃