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How to Prepare Digestion Buffer

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4 replies to this topic

#1 mike90

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Posted 07 February 2012 - 11:00 PM

Hello guys.. I need your help.. I'm going to prepare digestion buffer before DNA extraction. I'm working with cancer cell line.
This is the list of reagents:


100 mM NaCI
10 mM Tris.CI, pH 8.0
25 mM EDTA, pH 8.0
0.5 % SDS
0.1 mg/ml proteinase

* the final volume should be 1 ml.. can anyone show me the correct steps to prepare each of the materials?

i really appreciate your kindness... Thank you very much.

#2 Papaver

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Posted 08 February 2012 - 12:40 AM

I would prepare a stock solution of your buffer and reagents. Make solutions of 1 M Tris-HCl pH 8.0, and 0.5 M EDTA pH8, autoclave them; 20 % SDS. Then you can prepare your basic buffer, e.g. 100 ml: 0.58 g NaCl, 1 ml Tris-solution, 5 ml EDTA solution, 2.5 ml SDS, fill up with water.
When preparing the digestion add fresh proteinase to the desired volume.

#3 Don8

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Posted 12 October 2014 - 01:36 AM

Hello!!! I need your help :) How to prerare solution of 50 ml: 

guanidine isothiocyanate 3M

Tris-HCl 20mM

Triton X-100 2%

pH 7.0

 

I have stock solution guanidine isothiocyanate 3M.

 

Thank you very much.



#4 phage434

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Posted 12 October 2014 - 09:16 AM

You can't use your 3M GuIC solution, since you need the 3M final concentration.

 

On the other hand, you can come close to 3M this way:

1 ml Tris-HCl pH 7.0 1M solution

1 ml Triton X-100

48 ml 3M GuIC

 

Which will give you close to 3M GuIC, but a little less.

 

Otherwise, you'd need to work with a different GuIC stock (probably solid).



#5 Don8

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Posted 13 October 2014 - 12:07 PM

Thank you very much phage434!!!  I made the same calculations smile.png Can I rely on you for help?

My best regards smile.png







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