Preparation of 6% EDTA as Anticoagulat? - tell me how to prepare it........... (Jul/06/2007 )
PLZ tell me how to preapare 6% EDTA as anticoagulat?????
dissolve it in distilled water or double distilled water
use heat and stirer to dissolve it???????
and how to prepare 20% EDTA in murine PBS
what type of EDTA used in each recipe???????
and which of them suitable for blood obtained by retro-orbital bleeding from rats
Thanks very much. Best regards.
6% EDTA is pretty high. (Human) Blood is usually collected into 0.1% solution. You prepare these tubes by adding the 0.1%solution of EDTA to the tubes then letting them evaporate dry at room temp. You can use either the disodium or the dipotassium salt (dopotassium being more soluble).
20% EDTA in PBS is even higher. The 20% probably means w/v (weight by volume). So into 100ml you'd add 20mg.
I'd probably try a range down to 15 or even 0.5%. Outcome measures could either be calcium concentration (easy spectroscopy using cresolpthelein) or perhaps the clotting time (not sure about this one). EDTA inhibits the clotting be sequestering calcium. It thus inhibits all calcium-dependent enzymes not just the clotting ones. Heparin is another anticoagulant that only inhibits the clotting. I imagine it depends what systems you are looking at as to which you use. Stick to what the literature uses and if neither have been reported...try both. If you get something interesting get human samples, repeat the experiment and publish in a clinical lab journal. (Then collect Nobel Prize).
Hope that helps.
that would be 20gm.
20% EDTA in PBS is even higher. The 20% probably means w/v (weight by volume). So into 100ml you'd add 20mg.
I'd probably try a range down to 15 or even 0.5%. Outcome measures could either be calcium concentration (easy spectroscopy using cresolpthelein) or perhaps the clotting time (not sure about this one). EDTA inhibits the clotting be sequestering calcium. It thus inhibits all calcium-dependent enzymes not just the clotting ones. Heparin is another anticoagulant that only inhibits the clotting. I imagine it depends what systems you are looking at as to which you use. Stick to what the literature uses and if neither have been reported...try both. If you get something interesting get human samples, repeat the experiment and publish in a clinical lab journal. (Then collect Nobel Prize).
Hope that helps.
i am dealing with blood of rats not human. i see that it is very sensitive and coagulated very quickly
if you are starting with EDTA (acid), you need to reduced the pH down to about pH8. At pH 7, EDTA is quite insoluble at the concentrations proposed. So start with EDTA powder mixing in water and throwing in NaOH pellets to increase the pH.
so i should add NaOH pellets until EDTA is being soluble in water???????
no nedd to heat and using magnetic stirer??????????????????
i want to know should i used distilled H2O or double distilled H2O?????????????
Best regards
well, even at pH 8 EDTA doesn't go immediately into solution (at the concentrations here). So make up the EDTA solution, then use a pH meter to adjust the pH to pH8.
A magnetic stir bar is a must. Although, you don't need heating.
"distilled H2O or double distilled H2O" in modern context, has no difference. We don't actually distill water anymore, all labs I have seen use reverse osmosis to purify their water. miliQ water.
I guess one should use something good enough for PCR (which you might do later). So use the purest form you can. You will however have to autoclave your EDTA solution after making it. The EDTA powder and equipment used to make the solution isn't sterile.