Comet assay sperm cells - (Sep/13/2012 )
greetings i am new here, and i'm sorry if this is not the correct place but if someone can help me lighting up my path, i'm just a student in biology but i'am helping one of my teachers (well,i'm very newbie, so may be he is helping me) , and i'm trying to figure out the use of some substances (my teacher knows, but he says "figure it out by yourself, then i will ask you") in 2 Lysis solutions from a protocol for comet assay that i got, the first lysing solution:
reactive: use for maybe?
*NaCl : osmotic shock i guess.
*EDTA: avoids the degradation of DNA while the preparation and in the electrophoresis run.
*DMSO: protects the cells
*triton X100: lyse the cells
at 4°c, may be (i think...again) is for inhibits the enzymes from the acrosome from the sperm cell (proteases. hyaluronidase...etc)
well i know that all the lysing solutions are made for lyse the cells, and normally the compunds in them are salts and detergents, but reading i think that is the use of them.
the second lysing solution
NaCl:the same osmotic shock?
Na2EDTA:is obvious a detergent but, i don't know...
tris:protects the DNA from the pH changes.
proteinase K:inhibits the DNAases, and other enzymes.
DTT:protects the cells from oxidation damage
at 37.7°c, i think is for the proteinase K.
well, if someone can help me, thanks in advance, and sorry for my bad english...
Part 1- NaCl - for osmotic shock you would need a concentration that is above or below the normal physiological range - Normal saline is 150 mmol/l or 0.9%.
EDTA: chelates metal ions, which helps inhibit those enzymes that have a metal co-factor.
DMSO: This one is tricky - it's actually to scavenge free radicals The protectant function only comes into play at really low temperatures where it prevents ice crystal formation.
Triton X-100 is a detergent, used to pop the membranes.
Part 2:NaCl and EDTA are the same as above. EDTA is not a detergent
Tris: buffering is correct. Tris buffers strongly at pH7 or there abouts.
Proteinase K: OK.
DTT is there to denature proteins, and probably to help prevent secondary structure in the DNA.
37 is the optimal temperature for proteinase K.
thank you very much, it was very helpful for me!
well everithing is going good in the investigation of my professor but (until) a co-worker fill the first lysis coplin with just DMSO and triton X 100 (the rest is just dH20) , but i just wonder, will the comets appear? the second one is full, so the osmotic shock will appear because of the Na2EDTA and the NaCl? i am pretty anxious for her mistake, because we will need to search for new samples... and thats quite impossioble right now, we need results by the next week, i'm sorry for my bad english.
I think the cells will be too damaged by the water for you to see clear comets.
I had to re obtain the samples again, but all went well thanks for your help bob1!