I've been doing the extraction and precipitation for 3 times already and I didn't get any pellet (Nanodrop result also didn't show that there's DNA in the solution) Here's a protocol of what I've done.
1. Increase volume/tube to 300µl by adding Milli Q water (there're DNA 5 micrograms/tube)
2. Add 300µl (1:1) of phenol-chloroform (saturated in TE buffer)
3. Heavily shake the mixture for 30 sec (hand shake)
4. Full speed spin down at RT for 2 min
5. Transfer supernatant to a fresh tube
6. Repeat step 2-5 but add chloroform instead of phenol-chloroform
7. Add 30µl of 3M sodium acetate (NaAc) pH 5.5 and 900µl of cold 100% EtOH and mix well
8. Incubate the mixture on ice for 10-30min or at -20C overnight (I tried both and still didn't get the pellet)
9. Full speed spin down at 4C for 30 min
10. Discard supernatant. Then add 1ml of cold 70% EtOH
11. Spin down 15-20 min at 4C full speed
12. Discard supernatant and air dry the pellet
13. Resuspend the pellet in 10µl of RNase-free water
Before starting this extraction/precipitation, I did restriction enzyme digestion to the plasmid I got from Midiprep and run the product on gel. Everything worked nicely. When I add NaAc and EtOH to the mixture (step 7) I saw bubbles occuring in the tube as well (is that a proof that I have DNA in there?). I also tried spinning down again (repeating step 9 2-3 times), incubate the mixture on ice for 20 minutes, and/or at -20C overnight.
I kinda suspect the 3M sodium acetate (pH5.5) I prepared. The total volume of 3M sodium acetate will be 50ml so I add 0.15x82.03 = 12.3g of sodium acetate (I used just sodium acetate, no anhydrate/trihydrate or anything like that) and fill it up with distilled H2O to 50ml (well, actually just 35-40ml leaving some rooms for acids to be added in). Is this calculation right?
I also wonder if I can use HCl instead of acetic acid in order to adjust the solution's pH to 5.5 (because this is what I did!). I know that, doing this, in the end I'll have NaCl and acetic acid in there, right? Does this somehow interfere the extraction or precipitation I'll need to do later on?
Now I'm totally blinded and can't figure out what was going on... please.. anything would be highly appreciate it.
Thanks so much everybody for your help!!!
Edited by missdaisy, 14 April 2012 - 07:35 PM.