Protocol Online logo
Top : Forum Archives: : Molecular Biology

Problem with anticoagulant for blood collection - (Apr/13/2007 )

PLEASE TELL ME WHAT THE PROBLEM CONCERNING MY ANTICOAGULANT

i collect blood from retro orbital site from rat using capillary filled with EDTA and the blood collected into tube containing amount of EDTA but the blood form red threads immediately and when i perform the lysis step using red cell lysis buffer those threads remain after several washing

EDTA which i use prepared as follow (9.3 grams of EDTA disodium and 1.12 grams of NaOH dissolved in 50 ml double distilled water) it's 0.5 M concentration

please tell me where the problem? is it the anticoagulant????

-Jehane-

QUOTE (Jehane @ Apr 13 2007, 12:26 AM)
PLEASE TELL ME WHAT THE PROBLEM CONCERNING MY ANTICOAGULANT

i collect blood from retro orbital site from rat using capillary filled with EDTA and the blood collected into tube containing amount of EDTA but the blood form red threads immediately and when i perform the lysis step using red cell lysis buffer those threads remain after several washing

EDTA which i use prepared as follow (9.3 grams of EDTA disodium and 1.12 grams of NaOH dissolved in 50 ml double distilled water) it's 0.5 M concentration

please tell me where the problem? is it the anticoagulant????



EDTA conc we use is 6%. Add EDTA solution to collection tube in accord with ratio blood\ Anticoagulant sol = 1\19 better to cold tube near 4C

And question - what do you want to prepare serum or isolate cells or other one? About serum preparation - don't worry about clogs. When we collect blood from retro orbital sinus of mice we used sterilized dry paster's pipette and collect into dry glass tube. Then incubate at 37 for 40 min and then 40 min at 4C after that good retraction occure. Then with pipette around the well in tube to dettache clot from inner space of tube than centrifuge for 10 min at 1200 RPM or 1500RPM in mini eppendorf centrifuge.

-circlepoint-

QUOTE (circlepoint @ Apr 13 2007, 02:14 AM)
EDTA conc we use is 6%. Add EDTA solution to collection tube in accord with ratio blood\ Anticoagulant sol = 1\19 better to cold tube near 4C

And question - what do you want to prepare serum or isolate cells or other one? About serum preparation - don't worry about clogs. When we collect blood from retro orbital sinus of mice we used sterilized dry paster's pipette and collect into dry glass tube. Then incubate at 37 for 40 min and then 40 min at 4C after that good retraction occure. Then with pipette around the well in tube to dettache clot from inner space of tube than centrifuge for 10 min at 1200 RPM or 1500RPM in mini eppendorf centrifuge.


Thank You very much.
i want to prepare DNA extraction from blood ready for PCR.

PLEASE tell me what do you mean by 6% EDTA. how do you prepare it? and what ite PH valu????
AND do you use EDTA or EDTA disodium?????

Best Regards.

-Jehane-

PLEASE TELL ME WHAT THE PROBLEM CONCERNING MY ANTICOAGULANT

i collect blood from retro orbital site from rat using capillary filled with EDTA and the blood collected into tube containing amount of EDTA but the blood form red threads immediately and when i perform the lysis step using red cell lysis buffer those threads remain after several washing

EDTA which i use prepared as follow (9.3 grams of EDTA disodium and 1.12 grams of NaOH dissolved in 50 ml double distilled water) it's 0.5 M concentration

please tell me where the problem? is it the anticoagulant????

-Jehane-

Jehane, we use EDTA taken from blood sampling tubes (like the one's in the hospital- EDTA-tubes (Vacutainer system). No problem with DNA extraction!

K.


PS: Please, don't post every question in three or more different forums. Most members around use the new posts button to see new posts...

-kr├╝melmonster-

QUOTE (Jehane @ Apr 13 2007, 03:14 AM)
QUOTE (circlepoint @ Apr 13 2007, 02:14 AM)
EDTA conc we use is 6%. Add EDTA solution to collection tube in accord with ratio blood\ Anticoagulant sol = 1\19 better to cold tube near 4C

And question - what do you want to prepare serum or isolate cells or other one? About serum preparation - don't worry about clogs. When we collect blood from retro orbital sinus of mice we used sterilized dry paster's pipette and collect into dry glass tube. Then incubate at 37 for 40 min and then 40 min at 4C after that good retraction occure. Then with pipette around the well in tube to dettache clot from inner space of tube than centrifuge for 10 min at 1200 RPM or 1500RPM in mini eppendorf centrifuge.


Thank You very much.
i want to prepare DNA extraction from blood ready for PCR.

PLEASE tell me what do you mean by 6% EDTA. how do you prepare it? and what ite PH valu????
AND do you use EDTA or EDTA disodium?????

Best Regards.


FOR PCR you should work with blood(plasma). We take 3g EDTA disodium salt + 50ml H2O. Of course capillar should be pretreated with the same EDTA solution. When collect blood into tube mix well with anticoagulant well and put on ice. Centrifuge 20 min 3000 rpm RT. Venoject system or vacutainer is good but It was difficult for us to collect from ROS of mouse. It should be special training may be. Think about fasting collection blood for blood PCR

-circlepoint-

QUOTE (circlepoint @ Apr 13 2007, 02:14 AM)
EDTA conc we use is 6%. Add EDTA solution to collection tube in accord with ratio blood\ Anticoagulant sol = 1\19 better to cold tube near 4C


PLEASE tell me the correct ratio of blood/anticoagulant = 1/19 or 19/1

as if i will take 2 ml of blood i should use 100ul of anticoagulant
is it true??????????????????????????????/

-Jehane-

QUOTE (Jehane @ Apr 16 2007, 07:58 AM)
QUOTE (circlepoint @ Apr 13 2007, 02:14 AM)
EDTA conc we use is 6%. Add EDTA solution to collection tube in accord with ratio blood\ Anticoagulant sol = 1\19 better to cold tube near 4C


PLEASE tell me the correct ratio of blood/anticoagulant = 1/19 or 19/1

as if i will take 2 ml of blood i should use 100ul of anticoagulant
is it true??????????????????????????????/


Sorry Jehane!
Correct one : blood/anticoagulant = 19/1

-circlepoint-