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Transfection


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#1 autumn

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Posted 18 March 2005 - 04:16 PM

# Should I always split my cells at their mid log phase? Or does it only matter at transfection? How do I know that my cells are in the mid log phase or not too long in the plateau?

# Where should I prepare the media? In the laminar flow hood or on an opened table (risks for contamination?) ?

#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?

#Should I always have HCO3- in the medium and what for?




Thanks.

Edited by autumn, 19 March 2005 - 09:55 AM.


#2 fred_33

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Posted 21 March 2005 - 01:19 AM

Should I always split my cells at their mid log phase? Or does it only matter at transfection? How do I know that my cells are in the mid log phase or not too long in the plateau?


cells that are up to 50 % confluency and less than 80% are in their mid log phase

Where should I prepare the media? In the laminar flow hood or on an opened table (risks for contamination?) ?


media must be prepared in the laminar flow

If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?

You should wash your cells twice with PBS. you may dilute trypsin in PBS. Inactivate the trypsin by adding medium.

Should I always have HCO3- in the medium and what for?


usually, cells are in 5%CO2 air. This ensure a combination with the media to automatically form HCO3-. This ions give the pH stability and it's essential (but automatic) to have it in the media

Questions answered?

Fred




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