This is my first time error, usually I never get this problem. I made 3M Sodium acetate but do not know how much micro litres to add. Can you let me know. Right now my sample with Iso 2 propanol is in -20 degree.
Hi Viji,
I'm using human peripheral blood. I added equal volumes of PCI. I am correct so far. I just need to know that to trouble shoot the precipitation step they usually add 3M Sodium Acetate but how much volume I don't know. To precipitate DNA I added equal volumes of chilled Iso 2 propanol and now the total volume is 8 ml. I've kept it at -20 degree.
Hi Smita,
For DNA isolation from gram-positive bacteria, I use 2%CTAB cell lysis buffer followed by Phenol: chloroform: isoamyl alcohol (24:25:1) extraction.
For DNA precipitation I use 0.6 volume ice-cold isopropanol (240 μl isopropanol for 400 μl aqueous phase) and 1/10 volume of 3M sodium acetate (pH-5.2) (40 μl NaOAc for 400 μl aqueous phase).
I always get very good result.
2% CTAB: Lysis Buffer -25ml
CTAB - 0.5 g
sterile nanopore water - 14.45 ml
1M TRIS-HCl (pH8) - 2.5 ml
5M NaCl - 7 ml
0.5M EDTA (pH8) - 1 ml
b-mercaptoethanol - 50 ul (add just before using CTAB, under hood)
I think this will help you.
Best wishes...........
Thanks Mr. Das. That sure was helpful. I'll try adding 1/10 volume of 3M NaOAc
Yes, I've added 1/10th and kept for overnight incubation at -20. Thanks Mr. Myti
Greetings to all.
Hey! thanx a tons for the 3M Na acetate idea. It worked!! I got a pellet but now I must confirm with Nanodrop readings for my satisfaction :-)
Have a fabulous day!!
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