What can be the reason for getting black slimy precipitate on adding Proteinase K and Lysis Buffer to blood sample? Adding some more details : Blood was collected in pre-filled EDTA tubes and was snap frozen. thawed at room temperature, diluted with PBS. After adding Proteinase K and Lysis buffer, black slimy precipitate was obtained which could not be loaded on purification column since it coluld clog it.
Have you ever tried this protocol before without getting the black, slimy precipitate? If I had to guess, I would say that the red blood cells lysed, the hemoglobin was released, it was partially digested by the proteinase K and perhaps denatured by whatever is in the lysis buffer, exposing normally buried hydrophobic parts of the protein, causing the colored protein to aggregate and precipitate. Proteolysis may also have caused release of insoluble heme. I imagine it's like what would have happened if you had boiled the blood.
What is the goal of the procedure?
Hi Adam, thanks for your reply. The goal of procedure is DNA isolation from Blood. Can we assume this slimy precipitate as debris or partially clotted blood? Not sure of this but want to know if it could be this. Please clarify. Thanks, Neetika
Here is a web page concerning kits for isolation of DNA from whole blood.
https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/genomic-dna-extraction/blood-dna-extraction.html
And here is a review article about methods for DNA extraction from blood.
https://www.dovepress.com/methods-for-extracting-genomic-dna-from-whole-blood-samples-current-pe-peer-reviewed-fulltext-article-BSAM
Perhaps if you dig into the original literature on your method, you will be able to find out whether or not the slimy precipitate is supposed to happen.