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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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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 -80C

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 l ---------> RNA isolation: samples on ice!!

DNA isolation
- can be stored o/n 4C
- 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 4C
- 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 4C
- remove supernatant
- + 1 ml 70% ethanol (II)
- incubate 2-3' RT
- centrifuge 5' 2000 g 4C
- 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' 65C
- optional for large pellets:
- incubate o/n on rotating platform 4C
- centrifuge 10' >12000 g RT
- transfer supernatant to new tube
- discard pellet remaining after centrifugion
- store 4C

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?

-ien-

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.

-phage434-

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.

-mdfenko-

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?

-ien-

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.

-mdfenko-

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?

-HomeBrew-

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

-ien-

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

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?

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?

-ien-
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