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Method Article
Isolation method for human metaphase chromosomes
wenmao huang
commented on 18 December, 2019
Great protocol! I have one question that what is the Percoll buffer (see REGENT SETUP), did not find the details. And what is polyamine stock solution for?
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This is a preprint. It has not completed peer review.
Method Article
Isolation method for human metaphase chromosomes
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 13 Aug, 2008
Community comments: 2
Metrics
Comments: 2
HTML Views: ...
wenmao huang
commented on 18 December, 2019
Great protocol! I have one question that what is the Percoll buffer (see REGENT SETUP), did not find the details. And what is polyamine stock solution for?
View 1 reply
Kayoko HAYASHIHARA
replied on 23 December, 2019
Thank you very much for your interest. We are sorry that we forgot to post reagent setup. Please see below. REAGENT SETUP >PA buffer Mix 50 mL of 10× PA buffer stock solution, 50 mL of 10× EGTA solution and 600 mg of digitonin (SIGMA Aldrich Corp.), and adjust the volume to 500 mL followed by shaking at 37°C for 40 min. Centrifuge the buffer at 3,000 rpm and at 4°C for 5 min followed by filtration through the filter with 0.45 μm pore size on ice. Adjust pH to 7.2 with HCl after adding 250 μL of polyamine stock solution, 500 μL of 100 mM PMSF solution and 500 μL of 2-mercaptoethanol (Nacalai Tesque, Inc.). >Wash buffer Mix 15 mL of 20× wash buffer stock solution, 3 mL of thiodiglycol (SIGMA-Aldrich Corp.), 300 μL of 100mM PMSF and 500 μL of 30% Empigen BB (CALBIOCHEM), and adjust the volume to 300 mL and pH to 7.4 with KOH. >Percoll buffer Mix 89 mL of Percoll (GE Healthcare UK, Ltd.), 5 mL of 20× wash buffer stock solution, 1 mL of thiodiglycol, 100 μL of 100 mM PMSF, 166 μL of 30% Empigen BB and 200 μL of polyamine stock, and adjust the volume to 100 mL and pH to 7.4 with KOH. >8% paraformaldehyde/XBE2 Dissolve 0.8 g PFA in ~8.5 mL of MilliQ at 60°C keeping pH around 7~8 with NaOH. Add 1 mL of 10× XBE2 and adjust the volume to 10 mL. Dispense the solution into tubes with ~500 μL aliquot and freeze them until required.
Kayoko HAYASHIHARA
replied on 23 December, 2019
Thank you very much for your interest. We are sorry that we forgot to post reagent setup. Please see below. REAGENT SETUP >PA buffer Mix 50 mL of 10× PA buffer stock solution, 50 mL of 10× EGTA solution and 600 mg of digitonin (SIGMA Aldrich Corp.), and adjust the volume to 500 mL followed by shaking at 37°C for 40 min. Centrifuge the buffer at 3,000 rpm and at 4°C for 5 min followed by filtration through the filter with 0.45 μm pore size on ice. Adjust pH to 7.2 with HCl after adding 250 μL of polyamine stock solution, 500 μL of 100 mM PMSF solution and 500 μL of 2-mercaptoethanol (Nacalai Tesque, Inc.). >Wash buffer Mix 15 mL of 20× wash buffer stock solution, 3 mL of thiodiglycol (SIGMA-Aldrich Corp.), 300 μL of 100mM PMSF and 500 μL of 30% Empigen BB (CALBIOCHEM), and adjust the volume to 300 mL and pH to 7.4 with KOH. >Percoll buffer Mix 89 mL of Percoll (GE Healthcare UK, Ltd.), 5 mL of 20× wash buffer stock solution, 1 mL of thiodiglycol, 100 μL of 100 mM PMSF, 166 μL of 30% Empigen BB and 200 μL of polyamine stock, and adjust the volume to 100 mL and pH to 7.4 with KOH. >8% paraformaldehyde/XBE2 Dissolve 0.8 g PFA in ~8.5 mL of MilliQ at 60°C keeping pH around 7~8 with NaOH. Add 1 mL of 10× XBE2 and adjust the volume to 10 mL. Dispense the solution into tubes with ~500 μL aliquot and freeze them until required.