I extracted DNA from bacteria by classical approach or protocol phenol/chloroform method. Is it possible that this DNA is inactivated after extraction? If yes, which is the component that has disabled this DNA during extraction and how can we reactivate it?
"DNA is inactivated"
What does that mean?
If you refering to the traces of phenol, then there are 2 options:
a) Use phase lock tubes for phenol steps.
b) after dissolving DNA in water/10mM Tris, put them at 50 degree (open lid) for 10 minutes. Phenol traces will be gone.
even traces of phenol can inhibit a PCR reaction. Be very careful when you take the aqueous layer. Do not touch the interface with the tip.
"DNA is inactivated"
What does that mean?
If you refering to the traces of phenol, then there are 2 options:
a) Use phase lock tubes for phenol steps.
b) after dissolving DNA in water/10mM Tris, put them at 50 degree (open lid) for 10 minutes. Phenol traces will be gone.
The "Maniatis" protocol for removing phenol/chloroform is to treat the solution with an equal volume of ether equilibrated with water, heat to evaporate the remaining ether and then ethanol precipitate. However, oxidized phenol can damage the DNA.
I agree partly with Albino. 1) if your phenol is oxidized (red color) is can damage the DNA. The DNA cannot be repaired if this happens. Purchase new phenol, and perform the extraction again. Be sure to store phenol in a bottle protected from light at 4°C. It is also more stable at lower pH (pH 6-7), however we regularly equilibrate our phenol at pH 8 (with Tris-HCl) and do not have problems. 2) It is important to remove phenol after a phenol/CHCl3 extraction. However, I would NOT use ether. Instead, use CHCl3. Ether is a much bigger hazard in the lab as it is very combustible and volatile, and much more soluble in aqueous solutions than chloroform. Perform 1 or 2 extractions with chloroform after your phenol/chloroform extractions, and then ethanol precipitate. As suggested by Bharat, even a little phenol can inhibit most downstream reactions.
Inactivated DNA? I can just tell you that you have in the end purified DNA. Integrity of purified DNA you can check by gel electrophoresis. But inactivation - I don´t know what you mean with that.
thanks for your help. I mean that the DNA is damaged due to the extraction, i will consider your suggestions.
Yes during phenol chloroform method works on this principle of denaturing and renaturing the bacterial DNA; Plasmid DNA can recoil much quicker and the rest larger DNA which recoils slowly will be precipitated. But end of the process you will have your DNA renatured and thats how you use for the downstream applications. Check your protocol and the purity of DNA. Hope this helps good luck.
DNA extraction requires skill& remnants of Phenol can act upon DNA.Remove the Phenol layer carefully.U will not encounter this type of problem again.
Hi
Are you solve your extract DNA in buffer that having EDTA?
EDTA can chealate Mg+ and another ions that require for DNA-polymerase
Due to this subject DNA seem inactive in PCR and another reaction with ion requirement.
I used EDTA to solve the extract DNA . What can i use as substitute ?
I used tris-buffer ph=7-8 for solve, you can heating with your hands and little shake your vial for solving your DNA.
You can dissolve your DNA in 10 mM Tris-HCl pH 8.0 plus 0.1 mM EDTA. You may pipet up and down to recover any DNA that is stuck along the walls of the tube, then spin briefly to recover your solution.
DNA is an organic molecule, just like Taxol, for example. It has no catalytic function on its own so it's never really 'active' or 'inactive'.
If you mean you're having problems using your isolated DNA for downstream purposes, like PCR, restriction digestions, etc.. then it's unlikely that your DNA is problematic. More than likely there is some contaminant, like phenol, as others have suggested. It's recommended that you perform a number of downstream processing steps, like ethanol washes/precipitations, after a phenol:chloroform extraction of DNA to ensure that all residual phenol is removed.
Other contaminants may be problematic, like chelating agents (eg. EDTA) if you've used buffers containing them to redissolve your isolate. Precipitating the DNA and redissolving it in sterile miliQ grade water should fix this. If you isolate your DNA from a high starch tissue may also cause problems. You can remove starches using a 3hr Lithium Chloride precipitation.
All of these suggestions assume you've quality assured your DNA and demonstrated it isn't degraded of course. Running a gel to verify this would be my first step.
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