RBC LYSIS BUFFER - a good substitute of sucrose? - (Jan/23/2012 )
I've found this recipe for RBC lysis buffer (I'm extracting DNA from human blood):
0.32M Sucrose
5mM MgCl2
1% Triton X-100
10mM Tris-HCl, pH 7.6
But I need to adapt it to the reagents avaliable in my lab at the moment. I have no sucrose so what could be the best substitute? It seems that sucrose can increase osmolarity, separation of organeles and stabilization of the membranes... NH4Cl would do? I also don't have Triton so I'll use Nonidet....
Any suggestions would be much appreciated.
Thank you,
Andreia
Ammonium chloride would be a terrible replacement. You need a non-ionic molecule. Sorbitol is commonly used, but most other simple sugars would work. Glucose, e.g. Table sugar is pretty pure sucrose, btw.
This is what we use for RBC lysis, it's a different protocol.
10x LYSIS BUFFER (pH 8.0)
1.5 M NH4Cl
100 mM NH4HCO3
10 mM 0.5 M Na2EDTA
autoclaved
Trof on Tue Jan 24 17:49:55 2012 said:
This is what we use for RBC lysis, it's a different protocol.
10x LYSIS BUFFER (pH 8.0)
1.5 M NH4Cl
100 mM NH4HCO3
10 mM 0.5 M Na2EDTA
autoclaved
Yes we use that protocol too, for samples with 10ml of blood. But my supervisor wants us to optimize a new extraction protocol for samples with less volume of blood (5mL or less)...but thank you anyways
phage434 on Mon Jan 23 13:38:02 2012 said:
Ammonium chloride would be a terrible replacement. You need a non-ionic molecule. Sorbitol is commonly used, but most other simple sugars would work. Glucose, e.g. Table sugar is pretty pure sucrose, btw.
Yes that's what I thought too, I was tempted to get some table sugar and add it hehe...but since we have no sugars in the lab I was thinking about using a salt (I think the function of sucrose here is to increase osmolarity?). I also found this recipe weird because of the presence of Mg in the lysis buffer since DNAses use Mg...
Thanks for your input
Andreia Marques on Mon Jan 30 10:17:16 2012 said:
Yes we use that protocol too, for samples with 10ml of blood. But my supervisor wants us to optimize a new extraction protocol for samples with less volume of blood (5mL or less)...but thank you anyways
We hardly ever have 10ml, more around 3-6 ml. I don't understand what's the difference when using smaller volume of blood, you can always use less less buffer if you need to keep the ratio, or not?