How to Prepare Digestion Buffer - (Feb/08/2012 )
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.
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.
Hello!!! I need your help :) How to prerare solution of 50 ml:
guanidine isothiocyanate 3M
Triton X-100 2%
I have stock solution guanidine isothiocyanate 3M.
Thank you very much.
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).
Thank you very much phage434!!! I made the same calculations Can I rely on you for help?
My best regards