reprecipitate DNA - reprecipitate DNA (Sep/14/2008 )

hi admin and chatter,

As we know , If this ratio is 1.8 - 2.0, the absorption is probably due to nucleic acids.
A ratio less than 1.8 indicates that there may be proteins and/or other UV absorbers in
the sample, in which cases it is advisable to re-precipitate the DNA. A ratio higher than
2.0 indicates that the samples may be contaminated with chloroform or phenol and
should be re-precipitated with ethanol.

the question is if mine ratio less than 1.8 , what solution should i remove protein (reprecipitate DNA)

Sodium accete or Nacl


thanx

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3M sodium acetate

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What is the actual ratio? What are you planning on doing with the DNA, because it sometimes doesn't matter (other times it really does matter).

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What is the concentration of your sample?

We use 3M Na-acetate pH 5.2 for precipitation.

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i'm using method by sambrook,

1. 0.1 g sample homogenize with 500 ul disgestion buffer + Proteinase K + Rnase
2. incubate 55C overnight
3. then add 500ul PCI ; centrifuge
4. transfer aquas layer to the new tube
5. add 500ul PCI ;centrifuge
6. transfer aquas layer to the new tube
7. the add 500ul C:I ;centrifuge
8. transfer aquas layer to the new tube
9. add 1 ml ethanol 100%;centrifuge
10. remove aquas layer
11. add 500 ul 70% ethanol;centrifuge
12. remove ethanol and dried pallet
13. add 50 - 100 ul TE

when i do purify DNA using UV spectrophotometer (50 concentration) -always get below 1.7
and when i done in gel always still have protein

so i decide to add 3 M Na-acetate in number 9. as above step

As we know , If this ratio is 1.8 - 2.0, the absorption is probably due to nucleic acids.
A ratio less than 1.8 indicates that there may be proteins and/or other UV absorbers in
the sample, in which cases it is advisable to re-precipitate the DNA. A ratio higher than
2.0 indicates that the samples may be contaminated with chloroform or phenol and
should be re-precipitated with ethanol.

any idea? what should i do when the sample below 1.7

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This is my protocol for DNA precipitation. If the ratio is below 1.8, we precipitate the DNA and get the ratio up next time.

DNA Precipitation

The eluted DNA (50ul) is precipitated

Na Acetate (3M pH 5.2) = 5ul (1/10th )
Ethanol (100%,-20°C) = 130ul (2.5x); if total volume = 50ul

Vortex them throroughly.

Place in Dry ice for 2-5 min or
Place in –80°C Freezer for 2 hours or
Place in –20°C Freezer for overnight

Centrifuge for 30 min. at 4°C. Remove alcohol with out disturbing the pellet.

Add 200ul of 70% ethanol (-20°C) and centrifuge for 5 – 10 min. at 4°C.

Pipette out all the remaining alcohol and

Centrifuge briefly for a few seconds and pipette out all the remaining liquid.

Add warm TE/water and wait for 2-5 min. and then vortex (briefly) and centrifuge

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thank for that;s protocol

i more think .. 50ul u mean 50ul (DNA + TE) or should i remove the TE first.. and one more thing 1/10th ? u mean 1:10

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Don't place the proteinase with the rnase, you are loosing the rnase also the rnase work at 37

In general for organic extraction:
homogenize the sample with the buffer and protease or proteinase and incubate at 55-60C for a such small sample and 1h will be enough, let it cool down to 37C and add rnase incubate at 37C for 15 min up to 1h.
1 vol phenol (TE saturate) mix and centrifuge transfer aquous layer
1 vol phenol sevag (phenol/chloroform isoamyl) centrifuge tranfer aquous layer
1 vol chlorofom isoamyl centrifuge tranfer aquous layer
1 vol of TE to the last chloroform used so pick up if any DNA is left.
add 2.5 vol of absolute ethanol very cold (i store mine at -20C) plus 0.5 vol 3M Na Ac mix by invertion (the DNA thread should be seen) incubate at -20C overnight of -80C 1h (I prefer 1st)
centrifuge at max speed for at least 10 min.
decant etoh and add 0.5-1mL 70% etoh (RT) dislodge pellet (do not resuspend) centrifuge, repeat
decant the etoh and air dry or use a vacuum drier (savant). Do not over dry , resuspend in TE (0.1X-1X)

I don't use anymore the organic extraction (too toxic), instead of phenol/chlo. steps I add a solution of saturated NaCl (5M) and incubate in ice for 5 min, then centrifuge at high speed for 15 min all the proteins are in the pellet and I transfer the supernatant to a new tube (not touching the pellet or will get proteins) and add the etoh abs. and NaAc. etc...

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50ul of DNA solution, contains DNA dissolved in water or TE. Don't remove TE.

to 50ul add 1/10th volume (which is 5ul) of sodium acetate and 2.5x volume (which is 130ul ) of 100% ethanol.

Hopefully, this is clear.

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