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RBC LYSIS BUFFER - a good substitute of sucrose?


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#1 Andreia Marques

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Posted 23 January 2012 - 04:05 AM

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

#2 phage434

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Posted 23 January 2012 - 05:38 AM

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.

#3 Trof

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Posted 24 January 2012 - 09:49 AM

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

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#4 Andreia Marques

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Posted 30 January 2012 - 02:17 AM

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 :)

#5 Andreia Marques

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Posted 30 January 2012 - 02:21 AM

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 ;)

#6 Trof

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Posted 30 January 2012 - 02:50 PM

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 Posted Image

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?

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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