I have seen protocols which use between 50 and 100 ul/well of primary and secondary antibody. For Hoechst 200 ul/well.
These volumes can be varied? What would be the minimum volumes? I intend to observe the results using an immunofluorescence microscopy.
Thanks in advance
Dr. Moore is correct. As long as you samples are covered and don't evaporate. There are two more important considerations 1) The volume variation is higher with very small volumes (i.e. 10ul may vary 1-2 ul or 10-20% vs 100 ul may varying 1-2 ul 1-2%) 2) interassay/intraassay variability is lower with larger volumes. That being said, I have many developed assays with these plates using 20uL volumes.
Thanks for the answer Dr. Moore and Dr. Adams.
I want to use 10 microliter in 96 flat bottom well plate for some mixture screening. When i drop that 10 microliter sample in the well initially it remains in the center. But after a while it seems attaching to the edges of the well leaving center of the well kinda blank. I am trying to compare clarity/crystal growth/haziness of mixture of different ingredient putting them in different well in 10 microliter amount and comparing there absorbance. Although still i am getting some absorbance but i am not sure is it something accurate? Can it really differentiate between different wells as sample thickness is very very low?
Please let me know your thoughts and experiences.
Thanks in advance.
- Badges
- Science method