6 replies to this topic

[sirin]

Now I extract DNA from mammalian cells by
1.Lysis cells in Tris.Cl, EDTA, SDS, Pancreatic RNAasa 1 hr at 37 degree celcius
2.Add proteinase K activate at 55 degree celcius 1 hr
3.Extract by Phenol:Chloroform 2 time ,centrifuge and collect supernatant
4.Precipitate DNA by 3M sodium acetate 1/10 volume and 2.5V of absolute ethanol , keep in -20 degree celcius overnight
5.Centrifuge 12000 g 10 min then collect pellet , rinse with 70% ethanol
But I have so much salt in my pellet, when I run gel electrophoresis it has DNA in pellet
Can you advice me  "how to remove salt "
                           Thank you for your advice

[phage434]

The 70% ethanol wash is intended to remove salt.  You can do that more than once if you need to, since the DNA is insoluble in the wash.  Make sure you really have 70% rather than 95%, since the salt will not dissolve in 95% EtOH.

[sirin]

sirin, on 06 December 2010 - 08:09 AM, said:

Now I extract DNA from mammalian cells by
1.Lysis cells in Tris.Cl, EDTA, SDS, Pancreatic RNAasa 1 hr at 37 degree celcius
2.Add proteinase K activate at 55 degree celcius 1 hr
3.Extract by Phenol:Chloroform 2 time ,centrifuge and collect supernatant
4.Precipitate DNA by 3M sodium acetate 1/10 volume and 2.5V of absolute ethanol , keep in -20 degree celcius overnight
5.Centrifuge 12000 g 10 min then collect pellet , rinse with 70% ethanol
But I have so much salt in my pellet, when I run gel electrophoresis it has DNA in pellet
Can you advice me  "how to remove salt "
                           Thank you for your advice

Do you have any lysis buffer that no SDS because I think SDS and sodium acetate are interaction?
I rinse 3-4 times of 70% ethanol but it remain salt.
                                        THank you

[phage434]

I would add a chloroform only extraction before the ethanol precipitation.  You may be carrying over phenol into your sample.  I don't understand why you think there is salt in your sample.  What evidence is there for this?

[sirin]

phage434, on 06 December 2010 - 05:46 PM, said:

I would add a chloroform only extraction before the ethanol precipitation.  You may be carrying over phenol into your sample.  I don't understand why you think there is salt in your sample.  What evidence is there for this?

Could you explain "why you think phenol is cause of salt"
I attach picture before centrifuge
After that it has many salt

Attached Thumbnails

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[phage434]

It's hard to tell from only the picture, but I'm guessing that you are picking up protein and other junk from the phenol/chloroform extraction.  After you centrifuge, you should have a clear layer on top and a white layer in the middle, with (perhaps) a clear layer underneath.  You want to take just the clear top layer, avoiding everything else.  Don't try to get it all, at least for now.

It also looks as if you might have far too much sample for the amount of phenol/chloroform you are using.  You might scale the sample back by 10x and try again, or use a bigger tube and add more phenol/chloroform.

[sirin]

phage434, on 07 December 2010 - 05:11 AM, said:

It's hard to tell from only the picture, but I'm guessing that you are picking up protein and other junk from the phenol/chloroform extraction.  After you centrifuge, you should have a clear layer on top and a white layer in the middle, with (perhaps) a clear layer underneath.  You want to take just the clear top layer, avoiding everything else.  Don't try to get it all, at least for now.

It also looks as if you might have far too much sample for the amount of phenol/chloroform you are using.  You might scale the sample back by 10x and try again, or use a bigger tube and add more phenol/chloroform.

I will try again and be careful
                    Thank you for your advise