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how to get rid of DNA (in TE) contamination - protein and phenol removal (no kits please) (Jun/30/2009 )

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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?

-ien-

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

-mdfenko-

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 l TE buffer.
5. Add 17 l of 20% SDS (thus 0.5% v/v).
6. Add 2 l of a 20 mg/ml stock of proteinase K (thus 50 g/ml).
7. Mix by inverting several times, and incubate at 56C for two or more hours.
8. Add 600 l 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 l TE to the aqueous phase
12. Add 600 l 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 l TE to the aqueous phase.
16. Add 600 l 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 -20C.
20. Precipitate the chromosomal DNA for 3 - 5 minutes in a dry ice:ethanol bath, or for 15 - 30 minutes at -20C or -80C.
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 -20C 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 l to 200 l 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 65C; it is twelve times more active at 65C than at 25C. Rapid denaturation of the enzyme occurs at temperatures above 65C.

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!

-HomeBrew-

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?

-ien-

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?

-mdfenko-

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?

-ien-

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 File

-mdfenko-
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