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baaran

Member Since 01 Sep 2010
Offline Last Active Feb 23 2013 09:42 PM

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Posts I've Made

In Topic: A substitute for Triton-X100 in DNA extraction

17 November 2012 - 01:39 AM

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.

In Topic: A substitute for Triton-X100 in DNA extraction

17 November 2012 - 01:28 AM

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

In Topic: A substitute for Triton-X100 in DNA extraction

14 November 2012 - 02:29 AM

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?

In Topic: which ELISA-based or western blot-based apoptosis method to choose?

09 October 2012 - 01:32 AM

Thanks again, so I will go for DNA Fragmentation 1st and then Caspase 3 activity

In Topic: which ELISA-based or western blot-based apoptosis method to choose?

03 October 2012 - 01:22 AM

Curtis, on 30 September 2012 - 10:10 PM, said:

there are many different methods. I'll list them below

- CPE detection by AO/PI. this method is very easy and it's based on morphology change. You may need a fluorescent microscope.
- DNA ladder method. DNA fragmentation kits are commercially available, but you can also collect the dead cells and extract DNA yourself. You need to run the DNA on agarose gel and if it is fragmented it means the cells are definitely dead....this is the easiest explanation I can give.
- Annexin V/PI method, which is usually by flowcytometer and you don't have it
- caspase activity. This could be done by western blot or ELISA. you need to buy antibody against caspase-3 or 9 or 8. It depends really. They are commercial kits for this method too.

I would go for DNA fragmentation first.

Thanks for your Detailed Resonse
I wil choose DNA Fragmentation but
I have one more Question: Selection of Caspase is dependent on which factors? which one should I Select? 8?
thx again

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