5 replies to this topic

[Andreia Marques]

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

[phage434]

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.

[Trof]

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

[Andreia Marques]

Trof, on 24 January 2012 - 09:49 AM, 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

[Andreia Marques]

phage434, on 23 January 2012 - 05:38 AM, 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

[Trof]

Andreia Marques, on 30 January 2012 - 02:17 AM, 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?