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

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?

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.

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

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

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

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?

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

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

Or some kind of DNA-binding spin column like you'd use to exchange buffers -- maybe the Qiagen PCR clean-up type? Is this genomic DNA?

that's what fails horribly.....
It seems that the aqueous (TE) phase and the chloroform phase are inversed. I can pecipitate some DNA (only 10% of the input) from the lower phase and nothing from the upper phase.

Can you (or someone else) help me ..... troubleshooting

HomeBrew on Jul 20 2009, 02:40 PM said:

yes it is!
PCR clean-up seems a good idea (although I prefer to use no kits)
or can I use a QIAamp DNA kit?