Ethanol-fixation of Samples for Long-term Storage and Subsequent DNA Staining

-
I.Materials

70% Ethanol at – 20oC

DNA Staining Buffer:

Sodium citrate 0.25g
Triton–x 100 0.75ml
Propidium iodide 0.025g
Ribonuclease A 0.005g
Distilled water 250 ml

II Procedure

1. Place 1x106 cells from each sample into a polypropylene tube and centrifuge at 250 x g for 5 min.

2. Remove the supernatant as completely as possible without disturbing the pellet and add 1 ml of –20oC 70% EtOH dropwise to the cell pellet while vortexing.

3. Keep cells at -20oC until the day of DNA staining (cells can be stored for several weeks at -20oC).

4. On the day of DNA staining, take samples out of the freezer and spin them down by centrifugation at 250 x g for 5 min. Remove the supernatant as completely as possible without disturbing the cell pellet.

5. Add 1 ml of DNA staining buffer to the cell pellet and vortex gently and briefly. Keep cells for 15 min in the staining solution before acquisition on the flow cytometer.

Commercial sources:

Sodium citrate Cat# C7254 Sigma, St. Louis, MO
Triton-x 100 Cat# x-100 "
Ribonuclease A Cat# R4875 "
Propidium iodide Cat# 537059 Calbiochem, San Diego, CA

Comments

Post a Comment

Popular posts from this blog

Prepare 6X Laemmli buffer

-
Laemmli is a sample buffer to use in western blot. 6X sample buffer is added to each protein sample and is boiled or heated for 5-10 minutes. Recipe to prepare 10 ml: - 1.2gr SDS (sodium dodecyl sulfate) - 6mg bromophenol blue - 4.7ml glycerol - 1.2ml Tris 0.5M pH6.8 - 2.1ml dwater warm it a little bit and shake it till everything is dissolved. It takes time. - add 0.93gr DTT. Wait until it is completely dissolved. Aliquote and keep frozen at-20ºC.
Read more

0.1 M Triethanolamine

-
0.1 M TEA: TEA = Triethanolamine You will dilute TEA 1:10 in Millipore water: Pour 50 mL TEA into a 50 mL conical tube (TEA is very viscuous, so best to measure it in something that is disposable). Pour TEA from conical tube into 450 mL Millipore water with stirring. pH to 8.0
Read more

Laboratory Maintenance Checklist

-
Image
It is a known fact for anyone working in a laboratory be it the students, the researchers, scientists or lab assistants, it is necessary to follow safety rules and guidelines for their own safety. All laboratories have their lab safety symbols and lab safety equipments like goggles, mask, dress etc. At the same time, it is also very essential that a laboratory is well-maintained and taken care of so as to prevent any kind of dangers involved while working in a lab. As per rule, there should be routine maintenance and safety checks in a laboratory. Infact, it should be the duty of every person working in a laboratory to do a quick safety check after the completion of each experiment and before the starting of a new one. Or a responsible personnel should be assigned the duty to clean the laboratory and perform simple maintenance for lab care and safety . Laboratory Maintenance Presenting below few points which should be considered for laboratory maintenance: Bench:
Read more