I have been using DH10Bac competent cells for transformation of my recombinant DNA, I want to make those at lab instead of purchasing commercially every time. Can I use the normal procedure using Rubidium chloride or is there any different protocol?
Is there a specific benefit to the rubidium chloride protocol?
I prepare chemically competent E. coli of similar strains with a protocol very much like the one linked below (the OpenWetWare protocol). It's quick and easy.
http://openwetware.org/wiki/Preparing_chemically_competent_cells
I think, using Calcium chloride is easiest and fastest way to make your cell competent.
If you have an electroporator, you can make electrocompetent cells just by washing them in 10% glycerol(twice or thrice), reducing the volume and resuspending them again in 10% glycerol. It is an easy way but the no. of vials of competent cells you get the end is low. For example, I get only 8 vials of competent cells (about 50-70ul each) from 400ml culture of E. coli Dh5a.
THank you all for the reply. But DH10 Bac cells are different than DH5a cells. That is the reason why I was asking for a method to make them in the lab instead of buying them.
Hi Sai,
Please check out this link ( http://strubiol.icr.ac.uk/extra/baculovirus/bact_protocols.html#dh10bac ) for making home-made DH10Bac competent cells. Seems like that they are successful in making it.
There is nothing magical about DH10Bac in terms of making them competent, you can use any method that you usually use for competent cells, including making them electrocompetent. The strain is essentially DH10 with a plasmid in it.
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