DNA isolation - TRizol reagent (Apr/24/2007 )
Hi
I want to extract genomic DNA from cells to do PCR, I'm planning to use TRizol, can anyone explain me, why DNA is resuspended in NaOH??
and the protocol says that you should set pH after resuspention, but how do you do it if all you have is an ependorff tube with the DNA sample??
looking forward to your reply
Biotech
It is hard to redisolve DNA in water after the trizol reaction, it solves much better in a weak base (as NaOH in a low concentration, normally you'll use 8mM). For storage and for some downstream applications the basic pH can be a problem, so you'll need to correct it. Normally you do it blindly by just adding HEPES supplemented with 1mM EDTA according to a table like this one from Invitrogen:
pH Adjustment of DNA Samples Dissolved in 8 mM NaOH:
(For 1 ml of 8 mM NaOH use the following amounts of 0.1 M or 1 M HEPES, free acid.)
Final pH 0.1 M HEPES (μl)
8.4 ................. 86
8.2 ................. 93
8.0 ................. 101
7.8 ................. 117
7.5 ................. 159
Final pH 1 M HEPES (μl)
7.2 ................. 23
7.0 ................. 32
pH Adjustment of DNA Samples Dissolved in 8 mM NaOH:
(For 1 ml of 8 mM NaOH use the following amounts of 0.1 M or 1 M HEPES, free acid.)
Final pH 0.1 M HEPES (μl)
8.4 ................. 86
8.2 ................. 93
8.0 ................. 101
7.8 ................. 117
7.5 ................. 159
Final pH 1 M HEPES (μl)
7.2 ................. 23
7.0 ................. 32
ahh ok! thank you
something else, NaOH isn't it a strong base? it's just that when you use it at low concentration it is weak?
by the way is this a good extraction method? are there other protocols (none kits included.....I can't spend money on that)
bye
I used DNazol... about the same concept with Trizol. I personally skeptical about using NaOH to dissolve my DNA. I usually use TE buffer. But I guess it is ok once you added Hepes to neutralise the NaOH.
I guess the other method you can use is CTAB method. But you have to lyse the cells first. 
Of course kit will do the best job.
In my opinion, as lng as you don't have money for a kit, Trizol (or other Phenol:Chloroform extraction reagents) are the best choice.
K.
NaOH is a very strong base, you are right. But if a solution is very basic depends on the pH and 8mM NaOH shouldn't be too tough for your DNA... but TE is a possibility, too.
Hi, I used Trizol to extract DNA from cells and I resuspended it in NaOH 8mM as the protocol says, but it doesn't resuspend, I keep having a pellet floating around!!
What can I do??? Can I warm it at 45ºC to see if it slowly disappears?
help please!!
bio
you can briefly heat at 65° for 10' max.
If yit's not working, you may denature an aliquot as in PCR at 95° for 1' cool on ice.