9 replies to this topic

[baaran]

Hi all
I wanna do DNA extraction BUT I couldnt find Triton-X100
I know that there are some other substances that we can use for DNA extraction... BUT what are they? what are limitations of every substance like SDS or Tween?
Thx in advance

[hobglobin]

I use SDS usually and it has the advantage of not only dissolving proteins and lipids but denatures proteins.

[baaran]

hobglobin, on 12 November 2012 - 09:30 AM, said:

I use SDS usually and it has the advantage of not only dissolving proteins and lipids but denatures proteins.

SDS?
so u dont use proteinase k or DNA extraction kits?
can u send ur Extraction protocol for me?

[hobglobin]

I use Proteinase K and SDS together (but don't store them toegetehr in the buffer but add the enzyme when extraction the DNA). Anyway this enzyme is stable enough and the activity even improved with SDS:
See e.g. here or here. As far as I know only the concentration of SDS shouldn't be too high (0.5% in lysis buffer is a recommended concentration).

[lab rat]

Here are links to two resources about detergents used in molecular biology:

http://www.applichem...etergenzien.pdf
http://www.emdmillip...3117.ProNet.pdf

I have used SDS like Hobglobin,TX-100, and Tween-20 for different extractions. I think I used 1% of Tx-100 or 0.5% of Tween in my solutions.

ed:Sorry, my memory was a little off.

Edited by lab rat, 14 November 2012 - 11:24 AM.

[baaran]

hobglobin, on 14 November 2012 - 07:41 AM, said:

I use Proteinase K and SDS together (but don't store them toegetehr in the buffer but add the enzyme when extraction the DNA). Anyway this enzyme is stable enough and the activity even improved with SDS:
See e.g. here or here. As far as I know only the concentration of SDS shouldn't be too high (0.5% in lysis buffer is a recommended concentration).

So u mean I can use SDS alone or I cant?
I dont have TritonX100, Proteinase k or any Extraction kit... I have just SDS and Tween

[baaran]

lab rat, on 14 November 2012 - 11:16 AM, said:

Here are links to two resources about detergents used in molecular biology:

http://www.applichem...etergenzien.pdf
http://www.emdmillip...3117.ProNet.pdf

I have used SDS like Hobglobin,TX-100, and Tween-20 for different extractions. I think I used 1% of Tx-100 or 0.5% of Tween in my solutions.

ed:Sorry, my memory was a little off.

Thx for the files
ur memory is a bit off but mine is completely off!!!
so I should not expect u to tell me abt the detail of ur extraction protocol.

[hobglobin]

My protocol is more or less like this:
http://openwetware.o...ng_Out_protocol

Never tried out if not adding proteinase k works too...proteins (esp. nucleases and nuclear envelopes of DNA) are broke down by SDS or inactivated by the EDTA, so DNA will be released from DNA binding proteins and enzymes are not working. So DNA is protected against degradation too and is free.
Anyway my guess is that the yield will be reduced and you'll have more debris because proteins are not digested. This may co-precipitate more DNA (carrying it along), when the proteins are precipitated and later centrifuged.

[memari]

This is a home made recipe:
Prepare this Lysis buffer (Chip Lysis Buffer):

50 mM Tris, pH around 8.0
10 mM EDTA
1% SDS


Then mix these:
50% of Phenol(pH around 7.8 )/Chloroform
25% of Lysis buffer (Chip Lysis Buffer)
25% of Water

Now you can use it for DNA extraction:
Add 1 ml of that mixture above with pellet of bacteria or cells.
Dissolve bacteria or cells by pipetting up and down.
Keep vials on ice for 10 min.
put vials on vortexer horizontally and vortex for 15 seconds.
Centrifuge >11000 g for 15 min
Take supernatant.
Add the same volume of Isopropanol and invert several times and stay for 10 min at RT.
Centrifuge again. and wash pellet with 70-75 % Ethanol.


You can increase the SDS percentage to facilitate Cell and tissue disrupting.
And for RNA extraction:
http://babakmemari.w...e-and-protocol/
http://molecularbiol...php?f=2&t=19756

[lab rat]

Quote

Thx for the files
ur memory is a bit off but mine is completely off!!!
so I should not expect u to tell me abt the detail of ur extraction protocol.

Haha, I have no worries about yours.

I am assuming that you are doing animal DNA extractions? For animals, I did both PK and RNAse A. For bacteria and plants, I used CTAB extraction with no PK digest, although for plants I did add BME to the 2X CTAB and used chloroform:isoamyl extraction.