Comet assay sperm cells
Posted 13 September 2012 - 02:45 PM
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...
Posted 13 September 2012 - 04:50 PM
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.
I'm also shifting this to one of the homework forums...
Posted 15 November 2012 - 02:37 PM
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