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PLEASE HELP: phenol-chloroform extraction and EtOH precipitation problem


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#1 missdaisy

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Posted 14 April 2012 - 05:05 PM

Hi all,

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

Further information:
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!!!
Daisy

Edited by missdaisy, 14 April 2012 - 07:35 PM.


#2 phage434

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Posted 15 April 2012 - 07:39 AM

Some possible places for problems:
* Make sure you are adding the phenol-chloroform, not the buffer. It's the lower layer.
* Make sure you have no chloroform carried over when transferring from the chloroform wash
* It is very easy to miss the pellet. Orient the tube such that you know where to expect the pellet, and look for it after the first spin. With 5 ug, you should be able to see it.
* Make sure you don't dislodge and remove the pellet during the wash step.
* Make sure your 70% ethanol is really 70% still (and definitely not, for instance, 30%)

If you can find some glycogen or (better) Pellet Paint (Novagen) you can make the pellet larger and more visible. Usually it is losing the pellet that is the problem. I don't think your NaOAc preparation will be the cause of the problem.

#3 missdaisy

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Posted 15 April 2012 - 02:56 PM

Some possible places for problems:
* Make sure you are adding the phenol-chloroform, not the buffer. It's the lower layer.
* Make sure you have no chloroform carried over when transferring from the chloroform wash
* It is very easy to miss the pellet. Orient the tube such that you know where to expect the pellet, and look for it after the first spin. With 5 ug, you should be able to see it.
* Make sure you don't dislodge and remove the pellet during the wash step.
* Make sure your 70% ethanol is really 70% still (and definitely not, for instance, 30%)

If you can find some glycogen or (better) Pellet Paint (Novagen) you can make the pellet larger and more visible. Usually it is losing the pellet that is the problem. I don't think your NaOAc preparation will be the cause of the problem.


hi phage434, thank you very much for your answer and suggestion! :)
- I'm very sure I added the lower layer for the phenol-chloroform (I did add the upper layer in the first experiment I've done, but then I realized and added the correct one in my second experiment and so on.)
- After adding chloroform, shaking, and spinning down, I transfer just about 90-95% (approximately) of the upper layer solution into the fresh tube. Did I do it correctly? Maybe this is a stupid question, but how can I be sure that I have no chloroform carried over during the transfer?
- That's the problem I've got. I took the tube out of the centrifuge and looked for the pellet right away (after 30 min spin) but can't see the pellet 'at all'. :'( I also suspect my sodium acetate as I've mentioned so I've changed the solution to another lot that someone else has made. Still, I can't see any pellet in the tube..
- For the 70% EtOH, I have made the new 70% EtOH once again as well just to be sure I got a right percentage in there. (I made 100% EtOH 35ml + distilled water 15 ml) But... I don't think there is a problem about this solution since there was no pellet formed after adding salt and abs EtOH and the first spin..?

Additional question, I saw somebody mentioned in this site that if we saw the bubbles occurring in the tube after adding abs EtOH and NaAc, that means we have DNA in the tube, is that true?

Thank you so much again, seriously, for all your reply!

#4 phage434

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Posted 15 April 2012 - 06:14 PM

Make sure you mix well after adding the NaOAc and EtOH. Pellets are hard to see. Sometimes they are easier to see with no liquid in the tube, if you know where to look. My favorite way of spinning down after EtOH addition is to freeze at -80 for 1/2 hour, allowing the ethanol to gel. Then thaw and spin at the highest speed available for 1/2 hour. You might want to locate some DNA and spin it down in 70% ethanol just to see what a pellet looks like. You might want to up the concentration of EtOH slightly to start with -- 75 - 80% rather than 70%.

#5 missdaisy

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Posted 15 April 2012 - 06:18 PM

Make sure you mix well after adding the NaOAc and EtOH. Pellets are hard to see. Sometimes they are easier to see with no liquid in the tube, if you know where to look. My favorite way of spinning down after EtOH addition is to freeze at -80 for 1/2 hour, allowing the ethanol to gel. Then thaw and spin at the highest speed available for 1/2 hour. You might want to locate some DNA and spin it down in 70% ethanol just to see what a pellet looks like. You might want to up the concentration of EtOH slightly to start with -- 75 - 80% rather than 70%.



Thanks so much! You've been a great help! I'll try it that way this time :)




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