Hello everyone,
I isolated DNA from freshly taken Human blood but during processing I was getting RBC contamination in pellet .I washed with RBC lysis buffer 6-7 times followed by centrifugation but still got reddness in pellet while the supernatant was clear. So please suggest how to avoid this next time without using any kit? and I want to use this DNA as unmethylated control for methylation specific PCR. SO will that RBC contamination effect my results. I took 2 ml of blood initially and used 3 volume i.e 6 ml RBC lysis buffer for removin RBCs.
Hello, Lalita.
RBC lysis procedure is enough with adjusted spinning speed in your hands. Thank to RBC, there is no nucleus in the human mature RBC. The nucleus is kicked out during cell differentiation beginning from bone marrow in mammals. Birds, reptiles, and amphibians have nucleus in their RBC.
Do not worry about RBC any more.
DNA is usually bound to proteins and all dna isolation methods are designed to remove protein so a little globin from lysed red cells will make no difference and you will get good quality dna, Some methods use proteinase K and others salting out or fractionation between aqueous and phenolic reagents. If you really want to get rid of the red cells they are quite fragile and white cells are very robust so you can add water to the cell pellet for 30 secs and then spin down and most of the rbc will have lysed.
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