- → justwonder's Content
There have been 51 items by justwonder (Search limited from 21-February 19)
the haemocytometer takes up a fixed volume.
There are squares of different sizes reflecting a fixed fraction of the volume.
Depending on which squares you use to count the cells a multiplication factor is required. Put simply, method B uses the smaller squares for cell counting hence the larger multiplication factor, while in method A the large squares are used.
What do you think about the methods? Usually we take the average of the counted squares, but these methods don't do it. Any ideas?
1. Identify the clones to be expanded.
2. Using a sterile pipet tip and pipettor, resuspend the cells by pipetting.
3. Transfer 150 ml of the cell suspension one well of a pre-labeled 24 well plate.
4. NOTE: Use only the top row of each 24 well plate for new clones to allow room to expand the cells down the plate as they grow.
5. Add 2 ml of fresh medium to each well of the 24 well plate. Add 150 ml of fresh medium to each 96 well.
This is the protocol for transfering cells from a 96 to 24 well plate. The volume of each well in a 96 well plate is 150ml and i wonder what does it mean with point 3? How much should i transfer to a well of a 24 well plate? How much should i leave behind in a well of a 96 well plate?
and method B formula is; Cell concentration per milliliter = Total cell count in 5 squares x 50,000 x dilution factor. Where did he get 2500 in A and 50,000 in B?
Thanks for any inputs.
It is a normal reporter vector where the Poly-A sequence lays upstream of the promoter of the reporter gene. Any ideas?
what specific vector is this?
This is how the poly A sequence looks like. It lays upstream the SV40 promoter and is "used as transcriptional pause and for background reduction". What does it mean with that? Thanks.
So in this case the tube does not need to be sprayed, since it is already in the hood? Or should i spray it in the hood?
you better spray the package and open it into the hood. that is better than spray each tube. and once sprayed, the package can be kept in the hood.
I get different answers in this issue which makes me confused. So again, should i leave the UV light 24/7 when i don't work?
activate hood 15' before using it will ensure fresh sterile air; but you're right.10' of uv light will sterilize the hood.
Should i turn the blowers off when i have the UV light on?
Should i have the blowers on when i work?
Should i turn the blowers off when i don't work?
Thanks for any inputs which clare my mind.
Is that not more safety if i wipe the tube instead of the package since the tube is less than the package and if the package is already opened then the bacteria may come inside of it and if i only wipe outside of it then i can contaminate my cells if i open the package inside my hood? What do you think?
You should spray the package and then bring it into the hood and open it there.
So i have to turn on the UV light all the time except when i work? I thought i should leave the blowers on 24/7 and turn on and off the UV light after work and before work for only some minutes.
Turn on the blower 15 min before using it. After using it, turn off the blower and turn on the UV light.
# About the laminar flow hood; when should i turn off the blowers which circulate the air? After the work or should it be on all the time? Can i have it on at the same time with the UV light?
# Methylation usually happens in CG islets. Does it occur most on C or G?
# Why are HGPRT, TK and APRT amplified selection markers while neomycine and hygromycin are not amplified selection markers? Why do we use HAT medium for TK marker?
# Do I need mRNA destabilization signals for my gene expression? If so where should I add it?
# My book says; "cDNA are often obtained by addition of homopolymeric tails into the 5’ non coding region which should be removed.” Which homopolymeric tails are used for this purpose?
# Which sequences on the 3’ end that reduce mRNA stability?
So for these cell lines we usually use Simple media, which is Eagle’s MEM + serum?
established cell lines are cell lines that have been tested for many passages and don't change their characteristics (gene exprssion, morphology, morphotype...) They are quite old and their clturing conditions are well known.
What does it mean when it says in the context of selection of serum and medium: "Established cell lines- Simple media- Eagle’s MEM + serum" ? What are established cell lines?
Thanks for any ideas.
can you explain this? immortal cells are not resistant at the same time, are they? it would clear my mind alot. thanks.
i'm ok with that. you're right. but if your cells are resistant to puromycin whithout adding a vector with resistance marker, that mean that all your cells are immortal and in this case you do not need to add puromycin. you will do your selection after the transfection with the selective marker present on your vector.
in order to add geneticin or puromycin, should my cell have vector with marker which is resistant to these antibiotica? because the cells will die if it does not have resistant marker, won't it? or the cells are naturally resistant to these?
for freezing cells i use serul 10%DMSO or serum free medium +10%DMSO and it works very well. It's not necessary to add antibiotics like geneticin or puromycin.
you mean naturally resistant to and not that we have already transfected it with a resistant vector against these antibiotica?
But if you mean penicilin streptomycin by "antibiotics" you can add it in the medium after thawing. mammalian cells are normally resistant to these classical.
But NEVER add antibiotics during transfection