For the Lipofectamine 2000 protocol using X293T cells, how important is it to change the medium after the DNA-Lipofectamine complexes have been added to the cells? I read a protocol from Invitrogen which says "It is not necessary to remove the complexes or change the medium; however growth medium may be changed after 4-6 hours without loss of transfection activity." Has anyone proceeded without changing medium? Does that affect the end result?
yue is roght..LP2000 is highly toxix..
as 293T cells are not difficult to transfect i routinely use JET PEI it works great and it is easely home made and last for ages...
see https://www.researchgate.net/post/My_PEI_doesnt_work_at_pH_70
Do you use a home-made PEI?
We store it in aliquots at -20C. The vial in use is kept at 4C and works fine for a long time,
Here is our protocol how to make it:
PEI (1ug/ul) – PEI is Polyethylenimine 25kD linear from Polysciences (cat# 23966-2).
To make a stock solution:
• Dissolve PEI in endotoxin-free dH2O that has been heated to ~80°C.
• Let cool to room temperature.
• Neutralize to pH 7.0, filter sterilize (0.22um), aliquot and store at -20°C; a working stock can be kept at 4°C.
Hi Suhnrita,
Changing the medium is generally suggested to lower toxicity. One option would be to use biodegradable reagents such as DreamFect Gold
http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html
If you want a free sample let me know at [email protected]
Good Luck
Olivier
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