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justwonder's Content

There have been 51 items by justwonder (Search limited from 21-February 19)



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#13230 Medium-w/o GLUT?

Posted by justwonder on 30 March 2005 - 06:03 AM in Cell Biology

It says; "MEM, Earle's, DPM, autoclavable, w/o GLUT"

What does it mean with w/o GLUT? And are MEM, Earle's and DPM different types of mediums or the same medium? <_<

Thanks.



#13218 Cell counting

Posted by justwonder on 30 March 2005 - 03:35 AM in Tissue and Cell Culture

hi justwonder,

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.

Nick

<{POST_SNAPBACK}>



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?

Thanks.



#13183 Transferring cells from a 96 to 24 well plate

Posted by justwonder on 29 March 2005 - 08:46 PM in Cell Biology

Transferring cells from a 96 to 24 well plate:

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? :)


Thanks.



#13169 Cell counting

Posted by justwonder on 29 March 2005 - 04:18 PM in Tissue and Cell Culture

Can you read this link and tell me what you think of methods A and B? It does not make sense for me why method A formula is;Cell concentration per milliliter = Total cell count in 4 squares x 2500 x dilution factor
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? :)

http://www.animal.uf...macytometer.htm



Thanks for any inputs.



#13056 1 oz solute in 5 gal of water

Posted by justwonder on 27 March 2005 - 05:12 AM in General Lab Techniques

mY English is terribly bad, so please explain to me what is an oz and what is a gal when i have to dilute my solution as following:

1 oz solute in 5 gal of water . :D



THanks.



#13013 Seeding volume in cell culture flasks

Posted by justwonder on 25 March 2005 - 11:10 AM in Cell Biology

What are the typical seeding volumes you have for these T-25, T-75, T-150 flasks?



THanks for any ideas.



#12938 Poly-A sequences lay upstream of a reporter gene?

Posted by justwonder on 24 March 2005 - 07:56 AM in Molecular Biology

what specific vector is this?

<{POST_SNAPBACK}>

It is a normal reporter vector where the Poly-A sequence lays upstream of the promoter of the reporter gene. Any ideas? ;)


Thanks.

<{POST_SNAPBACK}>


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.

Posted Image



#12870 Poly-A sequences lay upstream of a reporter gene?

Posted by justwonder on 23 March 2005 - 12:37 PM in Molecular Biology

what specific vector is this?

<{POST_SNAPBACK}>

It is a normal reporter vector where the Poly-A sequence lays upstream of the promoter of the reporter gene. Any ideas? :D


Thanks.



#12841 Autoclaving and microwaving

Posted by justwonder on 23 March 2005 - 09:03 AM in General Lab Techniques

When the bottles don't have caps, can i cover them with folie and autoclave them or would this damage the folie?



Thanks.



#12827 Poly-A sequences lay upstream of a reporter gene?

Posted by justwonder on 23 March 2005 - 06:34 AM in Molecular Biology

Why should some Poly-A sequences lay upstream of a reporter gene? :(

Thanks.



#12826 Autoclaving and microwaving

Posted by justwonder on 23 March 2005 - 06:33 AM in General Lab Techniques

Should i put on the lids to my medium bottles when i autoclave them?

Should i cap my agar solution bottle when i microwave it?



Thanks.



#12814 Contamination issues

Posted by justwonder on 23 March 2005 - 03:38 AM in Cell Biology

hi
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.

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?

activate hood 15' before using it will ensure fresh sterile air; but you're right.10' of uv light will sterilize the hood.

<{POST_SNAPBACK}>

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?

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.



#12773 Contamination issues

Posted by justwonder on 22 March 2005 - 06:10 PM in Cell Biology

You should spray the package and then bring it into the hood and open it there.

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?

Turn on the blower 15 min before using it. After using it, turn off the blower and turn on the UV light.

<{POST_SNAPBACK}>

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. :P

Thanks.



#12767 Contamination issues

Posted by justwonder on 22 March 2005 - 05:43 PM in Cell Biology

# About sterile techniques; for example the package which is for the culture tubes. When i use one of these tubes, should i open the package outside the hood. Wipe the culture tube with EtOH before putting it on the hood or should i wipe the whole package with EtOH before putting it on the hood and open it to get the tube?

# 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?




Thanks.



#12764 more questions about protein expression...

Posted by justwonder on 22 March 2005 - 04:31 PM in Molecular Biology

on mammalian cells:

# 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? :P


Thanks.



#12763 protein expression...

Posted by justwonder on 22 March 2005 - 04:27 PM in Molecular Biology

in the mammalian cells:

# 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?



Thanks alot.



#12628 Thawing of competent bacterial cells

Posted by justwonder on 20 March 2005 - 06:46 PM in Microbiology

How can I thaw competent bacterial cells for transformation? Can i thaw the tube within my hand or would this damage my cells?



#12592 Tissue culture and cell culture?

Posted by justwonder on 18 March 2005 - 08:31 AM in Cell Biology

hi
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.

So for these cell lines we usually use Simple media, which is Eagle’s MEM + serum?



#12585 Tissue culture and cell culture?

Posted by justwonder on 18 March 2005 - 06:34 AM in Cell Biology

Thank you very much, badcell. I am doing my cell culture and here are some more questions related to it, so i hope that you guys can help me out. I think it is better that i keep these questions in this same thread. :unsure:


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.



#12583 Tissue culture and cell culture?

Posted by justwonder on 18 March 2005 - 05:28 AM in Cell Biology

What is the difference between Tissue culture and cell culture?

And when we talk about transformation in mammalian cells , does it mean that we transform the cells into cancer cells? :unsure:

Thanks for any ideas.



#12340 luciferase concentration?

Posted by justwonder on 15 March 2005 - 09:47 AM in Protein and Proteomics

thank you, badcell. you explained very well.



#12329 antibiotics in medium-WHEN?

Posted by justwonder on 15 March 2005 - 06:20 AM in Cell Biology

Thank you very much, Fred. You have helped me alot with this understanding. I could never do it myself. I truly appreciate your great help. :D



#12319 antibiotics in medium-WHEN?

Posted by justwonder on 15 March 2005 - 03:46 AM in Cell Biology

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.

:unsure: can you explain this? immortal cells are not resistant at the same time, are they? it would clear my mind alot. thanks.



#12309 antibiotics in medium-WHEN?

Posted by justwonder on 15 March 2005 - 02:41 AM in Cell Biology

thanks, Fred.

So in summary; add only antibiotica if the cells are naturally or artificially (vectors etc) resistant to them. Correct?



#12303 antibiotics in medium-WHEN?

Posted by justwonder on 15 March 2005 - 02:01 AM in Cell Biology

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.

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?

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.

you mean naturally resistant to and not that we have already transfected it with a resistant vector against these antibiotica?

But NEVER add antibiotics during transfection


i understood. :unsure:




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