2 replies to this topic

[Danny Chow]

I made a batch of compentent cell, and i calculated that the tranformation efficiency is about 4 x 10^5 cfu/microg DNA, is that good enough ? i am a beginner ...=__=

thanks

[phage434]

That's only good enough for subcloning already existing plasmids.  For cloning, you want 10^7 cfu/ug or greater.  You can make good cells; check here:
http://openwetware.o...competent_cells

[pDNA]

we get 10^8  cfu/µg electrocompetent cells by just washing them with ice-cold 1mHEPES ...the trick is to keep the cells cold (~2-4°C) all the time.

So everything must be coold in advance (centrifuge, eppis, ...really everything that comes in contact with the pellet) ...i prefer working in a 4°C room.

Pellet the cells, resuspend in ice-cold 1mM HEPES, wash pellet 2-4 times with 1mM HEPES, wash 1x with 10% Glycerin, concentrate Pellet 1000-fold by resuspending in 10% Glycerin, alliquote cells and immediatley quick-freeze in liquid Nitrogen. That's it!

Good luck!

Regards,
p