loose pellet in prep of electr-competent E.coli - inquire for a solution to solve the problem of loose pellet (Apr/27/2006 )
I have one question in the prep of electro-competent cell of E.coli:
whether you find the cells are easy to be dispersed after washing with ddH2O? (I spin at 4000g for 10min, in 4C)
This is my headache, every time I prepare the electro competent, I will lose 2/3 of the cell just
because the pellet is so loose that I pour a lot of cells out when decanting the supernatant.
I think that you can spin at twice that many rpm
thank you:) I will try that
I don't know whether increasing spinning speed will affect the efficiency of competent cell or not, so didn't dare to increase the speed before.
I agree with the following post. Also, in my experience if I start to decant and lose a significant part of the pellet, I will just repellet the cells and try again. You are bound to lose cells during the washing steps but should have a dense enough starting cell population for it to be negligible. Something else I found helpful is if you are going to use all the cells for transformation right away you can resuspended them in ddH20 instead of glycerol, which means you can leave some residual wash water in the tube and save more cells.
i would increase first the time and if it's not sufficent, you'll try more speed. 8000rpm will result in alteration of your cells. Moreover the resulting pellet will be crazy to resuspend for efficient washes...
so try 15' and if not enough 20' spins.