2 replies to this topic

[_THEEA_]

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      ... The problem is that I currently dont have small filter needed for sterilization of the buffer so I auto-calved it  . 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! 

 

Tea 

[mdfenko]

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.

[Zagami Francesco]

Purification of RNA from whole blood standard protocol : Five ml of whole blood is added to 20 ml of Red Blood Cell (RBC) Lysis Solution (0.15 M ammonium chloride, 0.001 M potassium bicarbonate, 0.0001 M EDTA, pH 7.2-7.4). The sample is mixed and incubated for 5 min at room temperature. The resulting RBC lysate is passed through two 47 mm diameter GF/D filters (Whatman glass microfiber filters - Grade GF/D - Aldrich cod. Z258245) to capture white blood cells and allow most plasma proteins and RBC contaminants to flow through the filters. The filters are washed with 20 ml of RBC Lysis Solution to further reduce contaminants. Nine ml WBC Lysis Solution (4 M guanidine thiocyanate, 1% Triton X-100, 0.05% sarkosyl, 0.01% Antifoam A, 0.7% beta- mercaptoethanol) are passed through the filters to release nucleic acids from the white blood cells, and the resulting lysate is collected comprising mostly RNA. Genomic DNA is retained on the GF/D filters and could be physically and/or chemically retrieved later. Four ml of water are passed through the GF/D filters to release additional RNA and this fraction is added to the WBC lysate. In the standard protocol, 13 ml of 80% sulfolane are added to the total lysate fraction for a final sulfolane concentration of 40%. The resulting mixture is passed over five GF/F filters (9.5 mm diameter each). The GF/F filters are washed three times with 2.5 ml of Low Salt Wash Solution (2 mM Tris (pH 6-6.5), 20 mM NaCl, 80% ethanol) for a total of 7.5 ml. The filters are air dried between each addition of Low Salt Wash Solution and after the final addition. The RNA is eluted from the GF/F filters with 100 microliters of RNase- free water. The eluted RNA is checked for yield and purity by UV spectrophotometry,