Please share your experience related to the stability of plasmid DNA at 37°C with respect to time (Hrs) in restriction digestion reaction? What is maximum time (Hrs) can we continue restriction digestion at 37°C without loosing stability of plasmid DNA?
Hi, Mangesh.
It will really depend on the restriction enzyme you're using for your digestion. In general ways, the longer you set your digestion reaction, the higher probability for the star activity to appear, but you can avoid this by using high fidelity restriction enzymes, setting shorter experiments or decreasing as much as possible the amount of enzyme (as they're usually kept in glycerol, which can promote star activity) if you actually want the digestion to last longer. For example, for my digestions I use BamHI-HF, that allows me to leave it up to 4h and without taking any risks of star activity. Besides non-desired cuts, if the reaction is contaminated with some other nucleases, they will be able to degrade the linear DNA produced by the digestion (since linear DNA is less stable than circular DNA). Based on my own experience, I'd strongly suggest that you follow manufacturer's instructions, purify your DNA after reaction (as the enzyme can remain attached to DNA) and check by running on an agarose gel.
I hope this will be of help!
Hi, Mangesh.
It will really depend on the restriction enzyme you're using for your digestion. In general ways, the longer you set your digestion reaction, the higher probability for the star activity to appear, but you can avoid this by using high fidelity restriction enzymes, setting shorter experiments or decreasing as much as possible the amount of enzyme (as they're usually kept in glycerol, which can promote star activity) if you actually want the digestion to last longer. For example, for my digestions I use BamHI-HF, that allows me to leave it up to 4h and without taking any risks of star activity. Besides non-desired cuts, if the reaction is contaminated with some other nucleases, they will be able to degrade the linear DNA produced by the digestion (since linear DNA is less stable than circular DNA). Based on my own experience, I'd strongly suggest that you follow manufacturer's instructions, purify your DNA after reaction (as the enzyme can remain attached to DNA) and check by running on an agarose gel.
I hope this will be of help!
Dear Mangesh
i completely concur with last answer. If your plasmid DNA is purified by either phenol / chloroform or by using columns then unlike RNA it is actually relatively stable and not prone to breakdown even at 37C.
The issue as already mentioned with restriction digestion relates to restriction enzyme specificity and not plasmid stability. Specifically for a few 100ng a 2 hour digestion will generally suffice and for greater than 1ug for complete digestion it is also not unknown to digest overnight.
That said as mentioned it is enzyme specific. Some enzymes more readily make non specific cuts - so called star activity - when plasmids are digested for more than 2 hours ( and certainly for overnight digestion). Check manufactures specifics for the enzyme you use: generally in the right buffer for up to 2 hours you shouldn't see non specific star activity; and moreover unless the manufacturer states that star activity is an issue it is generally acceptable and usually without problem to digest overnight
In most cases, high fidelity restriction enzymes are able to digest the 1ug plasmid within 15 minutes but we always keep it for 1 hr at 37°C. I tried the HF version of KpnI, BamHI, XbaI, EcoRI, NheI, ScaI, SacI, pstI, PvuI and go till 6 hrs at 37°C in recommended buffers to ensure the full digestion without affecting the stability of plasmid to minimize the chances of colonies with vector only. This digested plasmids work fine in cloning.
Thank you all - Juan J. Sanchez-Gil, Laurence Stuart Hall, Manoj Patidar.
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