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RBC lysis bufer please help


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#1 _THEEA_



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Posted 23 September 2015 - 11:58 AM

Please can someone help me with RBC lysis buffer for red blood cells?? I have prepared one and used it but it didn't work    sad.png sad.png ... The problem is that I currently dont have small filter needed for sterilization of the buffer so I auto-calved it unsure.png . I don't know if that could damage something I ajusted th pH after autoclave, but when I used the buffer with the blood it couldn't break the RBC and I wasn't able to preform RNA extraction. Here is the protocol that I sued to prepare my RBC lysis buffer:


 RBC lysis buffer 10x

1. Prepare 8,26 g of NH4Cl

2. Add 1,19 g of NaHCO3

3. Add 200 µL of EDTA [0,5 M, pH8]

4. Add a.d until 100 mL 5. Adjust pH to 7.3

6. Filter sterile 


I would appreciate any kind of help! 



#2 mdfenko


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Posted 24 September 2015 - 04:57 AM

the bicarbonate (and maybe the ammonium chloride) will decompose when heated in the autoclave.


also, you lost sterility when you adjusted the pH after autoclaving.


if you don't have sterile filters then you may be able to borrow from another lab.


however, if you prepare the buffer immediately prior to use (and, maybe, are able to filter any particulates) then you may be able to use it to lyse your rbcs without sterilizing.

talent does what it can
genius does what it must
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