17 replies to this topic

[ien]

after searching the archieves for at least an hour I wonder if anyone of you can help me

I used TRIzol to isolate DNA. After adding chloroform: large interphase (protein!?). Precipitation with ethanol gave large pellet. Ethanol washes (of course) did not help getting rid op excess of protein/cell debris. Pellet + TE (10:0.1) pH 8,0. Still large unsolved pellet, probably containing protein and cell debris. Unfortunately the tube still smells like phenol.

Does anyone know how to get rid of the protein/phenol and other contaminants...
Do I need to extract the DNA again from the TE?

Edited by ien, 30 June 2009 - 12:35 AM.

[mdfenko]

if you still have phenol present (after precipitation?) then you can remove it by extracting with chloroform. then reprecipitate.

[HomeBrew]

After a phenol extraction, we will extract again with phenol:chloroform:isoamyl alcohol, and then extract with chloroform until there is no white interface.  Here is our protocol for genomic DNA:

   1. Grow cells overnight in 5 ml of appropriate media.
   2. Fill one or more 1.5 ml microcentrifuge tubes with the overnight culture.
   3. Pellet the cells by centrifugation in a table-top microfuge.
   4. Decant the supernatant, and resuspend the pellet in 600 µl TE buffer.
   5. Add 17 µl of 20% SDS (thus 0.5% v/v).
   6. Add 2 µl of a 20 mg/ml stock of proteinase K (thus 50 µg/ml).
   7. Mix by inverting several times, and incubate at 56°C for two or more hours†.
   8. Add 600 µl buffer-saturated phenol to the tube. Mix by shaking.
   9. Centrifuge for 5 minutes in a bench-top centrifuge.
  10. Transfer the aqueous (top) layer to a fresh tube, avoiding the white interface layer. Discard the organic (bottom) phase.
  11. Add 100 µl TE to the aqueous phase ‡
  12. Add 600 µl phenol:chlorofom:isoamyl alcohol (25:24:1).
  13. Mix by inverting several times, then separate the phases by centrifuging for 5 minutes.
  14. Transfer the aqueous (top) layer to a fresh tube, avoiding the white interface layer. Discard the organic (bottom) phase.
  15. Add 100 µl TE to the aqueous phase.
  16. Add 600 µl chloroform.
  17. Mix by inverting several times, then separate the phases by centrifuging for 5 minutes.
  18. Repeat the chloroform extraction (above three steps) until no white interface remains §.
  19. Add 0.1 volume 3M sodium acetate to the final aqueous phase and fill the tube with 100% ethanol at -20°C.
  20. Precipitate the chromosomal DNA for 3 - 5 minutes in a dry ice:ethanol bath, or for 15 - 30 minutes at -20°C or -80°C.
  21. Recover the DNA by centrifugation. If even visible, the pellet will be clear, and may appear on the side of the tube.
  22. Carefully decant the ethanol and sodium acetate supernatant.
  23. Rinse the pellet once with -20°C 80% ethanol, and re-centrifuge.
  24. Carefully decant the 80% ethanol wash.
  25. Allow the pellet to dry at room temperature until no ethanol smell remains.
  26. Resuspend the pellet in 50 µl to 200 µl of TE buffer or sterile dH2O.


† Proteinase K is a nonspecific serine protease. It is not inactivated by metal ions, chelating agents (e.g. EDTA), sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. It is stable over a wide pH range (4 - 12.5), with optimal activity at pH 6.5 - 9.5. Activity can be stimulated by addition of denaturing agents (SDS and urea). The temperature optimum for the enzyme is 65°C; it is twelve times more active at 65°C than at 25°C. Rapid denaturation of the enzyme occurs at temperatures above 65°C.

‡ This and subsequent additions of TE serve to keep the sample size resonably high so you don't lose too much sample while removing the aqueous phase during each extraction step.

§ This usually requires 2 to 3 extractions.


Hope this helps!

[ien]

mdfenko, on Jun 30 2009, 06:14 AM, said:

if you still have phenol present (after precipitation?) then you can remove it by extracting with chloroform. then reprecipitate.

I've tried that.... no DNA present after precipitation (+1/10 vol NaAc pH 5.2 +2,5 vol etoh 100% nor with per 45 ul sample: +5 ul NaCl + 125 ul etoh 100%)

where's the DNA....?

[ien]

ien, on Jul 1 2009, 01:25 AM, said:

mdfenko, on Jun 30 2009, 06:14 AM, said:

if you still have phenol present (after precipitation?) then you can remove it by extracting with chloroform. then reprecipitate.

I've tried that.... no DNA present after precipitation (+1/10 vol NaAc pH 5.2 +2,5 vol etoh 100% nor with per 45 ul sample: +5 ul NaCl + 125 ul etoh 100%)

where's the DNA....?

the chlorofrom used was Chloroform:Isoamylalcohol (24:1) is that the problem?

[mdfenko]

ien, on Jul 1 2009, 04:29 AM, said:

the chlorofrom used was Chloroform:Isoamylalcohol (24:1) is that the problem?

no. it is fine (in fact, i would recommend it).

are you sure you had dna before re-extracting? maybe everything is still in the pellet?

why don't you give homebrew's protocol a try?

Edited by mdfenko, 01 July 2009 - 08:25 AM.

[ien]

the problem is that we have a few hundred samples of DNA in TE (10:0.1) pH 8.0.
nanodrop results vary between samples and for some samples the concentration increases over time, indicating still unsoluble DNA in the pellets of some samples (like you suggested).

the concentrations vary between 50 and >3000 ng/ul
the 260/280 ratio's vary between 1.69 and 2.39 (<1.8 = phenol and >2.1 = Protein contamination)
the 260/230 ratio's vary between 0.51 and 1.97

I just don't know where the DNA was after cleanup... And unfortunately we need this cleanup for some downstream applications.

Would you suggest to start at point 4 in homebrew's protocol or from point 4 straight to 8 etc.?
effectively that's almost exactly what I did.... the only difference is up to 400 ul TE in step 4 and 70% ethanol in step 24.

(Today I'm trying Tris-saturated phenol instead of PCI (25:24:1) in step 12. We'll see...)

[ien]

I forgot...
thanx for all suggestions so far!!

[ien]

It seems that the DNA is trapped in the chloroform phase instead of the aqueous phase. How can I tackle this problem?

[mdfenko]

with trizol the dna should be trapped in the phenol phase (trizol is acid pH and isolates rna in the aqueous phase).

you can release the dna into an aqueous phase by treating with base (the protocol says to use 8mM naoh).

in case you don't have or lost the protocol:

Attached Files

•  Trizol_Reagent.pdf   97.88K   105 downloads

[ien]

Homogenisation
- 1 ml TRIzol per 5-10x106 cells / 50-100 mg tissue
in 1,5 ml tube
cells/tissue in TRIzol can be stored at least one month -80ºC

Phase separation
- incubate 5' RT
- + 200 μl chloroform
- 15'' shake vigorously by hand
- incubate 2-3' RT
- centrifuge 15' 12000 g 4 °C
- lower red phenol-chloroform phase + white interphase -----------> DNA (+ Protein) isolation
- colourless upper aqueous phase ~550 µl  ---------> RNA isolation: samples on ice!!

DNA isolation
- can be stored o/n 4°C
- remove remaining upper aqueous phase (do not disturb white pellet)

DNA precipitation
- + 20 μl glycogen (1mg/ml)
- + 300 μl 100% ethanol
- mix by inversion
- incubate 2-3' RT
- centrifuge 10' 2000g 4ºC
- remove phenol-ethanol supernatant -------------> Protein isolation
label tube "D"
- short spin
- remove remaining phenol-ethanol

DNA wash
- + 1 ml 70% ethanol (I)
- mix by inverting until the pellet comes loose (at least 5 times)
- incubate 2-3' RT
- centrifuge 10' 2000g 4ºC
- remove supernatant
- + 1 ml 70% ethanol (II)
- incubate 2-3' RT
- centrifuge 5' 2000 g 4ºC
- remove ethanol
- short spin
- remove remaining ethanol

Redissolving the DNA
- air dry 5-15' RT
- + 100 μl TE buffer (10:0.1) pH 8,0
- incubate 10' 65ºC
- optional for large pellets:
- incubate o/n on rotating platform 4ºC
- centrifuge 10' >12000 g RT
- transfer supernatant to new tube
- discard pellet remaining after centrifugion
- store 4ºC

This is the protocol I used. Pellets were large and after addition of TE not completely dissolved. Also smelled like phenol.
Therefore I wanted to clean the DNA. I tried different options.
In essence: Chloroform:TE = 1:1, then precipitation (+1/10 vol NaAc pH 5.2 +2,5 vol etoh 100% or per 45 ul sample: +5 ul NaCl + 125 ul etoh 100%). No DNA present in upper (aqueous) phase. There was some (1/100 from input) DNA in the lower (chloroform) phase.

How can I tackle this problem?

[phage434]

If you need high throughput genomic DNA preps in small amounts, then I would look at the Zymo 96 well plate format kits.  They worked very well for us.

[mdfenko]

ien, on Jul 17 2009, 04:20 AM, said:

How can I tackle this problem?

you are close but not quite following the protocol. try following the protocol exactly as printed.

[ien]

mdfenko, on Jul 17 2009, 08:32 AM, said:

ien, on Jul 17 2009, 04:20 AM, said:

How can I tackle this problem?

you are close but not quite following the protocol. try following the protocol exactly as printed.

do you mean the sodiumcitrate washes? Or dissolving in NaOH?

the problem is that I used the protocol I mentioned earlier for several hundred samples. these samples are dissolved in TE and need cleaning-up (because of the phenol still present). Everything I tried failed (see earlier coments)....
So I understand that I need to change my protocol for new samples, but what to do with the old ones?

or did I misundertand your advice?

[mdfenko]

ien, on Jul 20 2009, 11:27 AM, said:

So I understand that I need to change my protocol for new samples, but what to do with the old ones?

or did I misunderstand your advice?

the only way that i am aware of to remove residual phenol is to extract with chloroform (or chloroform:iaa) and then precipitate.