Can DNA ethanol precipitation remove restriction enzyme? - (Aug/15/2006 )
I am going to perform some ligation and transformation reactions after restriction digest of my DNA samples, and thinking whether I can skip the heat inactivation process to kill all the restriction enzymes in the reaction, by doing ethanol precipitation of the digested DNA? So, can ethanol precipitation of DNA removes all the restriction enzymes in the reaction?
I need some confirmations so that I can stop worrying about enzyme residue ruin my downstream work.
Thank you very much in advance.
I precipitate my digests when I digest with 2 different enzymes (incomaptible buffers).
But if I were to gel purify them after the digest, few enzymes interfere with the further cloning steps, therefore only for those enzymes, i would heat inactivate before loading it on gel.
I'd do a phenol-chloroform extraction instead. This works fine.
At least the ethanol should also deactivate the enzymes, or not? Most restriction enzymes are claimed as soooo susceptible.
Nevertheless I heat deactivate them to be sure.
If you choose to heat-inactivate your RE, make sure to check the NEB website for the proper temperature to use. I typically PCI (25:24:1) extract and EtOH precip in order to get rid of my RE.
I've said this before elsewhere in the forums, but let me reiterate...I always, always, always gel purify my digested DNA (vector and insert) before ligation and transformation. This gets rid of uncut DNA, small pieces liberated by the digestion (if applicable), enzymes, CIP (if applicable), and is (IMHO) the single most important step you can take to insure a successful ligation and subsequent transformation.
I never heat inactivate anything, I never OD my samples (I don't even own a spectrophotometer that we can use for such a measurement), never obsess about vector:insert ratios, none of it.
I've been in this cloning racket for greater than twenty years, and rarely have a failed cloning reaction, mostly due to following this "gel-first" rule. The time you spend running the gel and recovering the fragments is miniscule compared to screening dozens (hundreds?) of vector-only transformants, or doing the cloning again and again because it fails repeatedly.
i use to pellet with ethanol the DNA after first enzyme.
But after the second one, i used to gel purify plasmid AND insert.
Now i use columns anytime it's possible. The problem is that ethanol does not remove proteins and it's a ligation failure condition, and i don't like phenol chlo purification, in which i lose DNA. Heat inactivation is case sensitive as it may not inactivate all enzymes and may damage the DNA ends of plasmid and or insert.
Moreover, with that, you don't get rid of the digested plasmid fragment.
I'm sure you've posted this elsewhere, but what gel extraction kit do you typically use?