How to increase the stability of the competence cells? We are using Rubidium chloride, transformation for competent cells is arguably the compound's most abundant use. I would like to know instead of rubidium chloride any other chemicals are there to prepare the competence cells.
Kindly let me know.
Thanks in advance.
You may consider buying commercial competent cells, with a transformation efficiency of > 10exp9/µg. You could also use electroporation. If you don't need highly competent cells (e.g. to transfer a plasmid for recombinant protein production into the production strain), CaCl2 competent cells are good enough and are the easiest to prepare and use
What you are describing sounds like Hanhan's method to me. This is one of the best methods to make competent with high efficiency. The use of Rubidium chloride was in the original formulation of the buffer, however, multiple derivatives exist. A good substitute would be MnCl2 as indicates in the CCMB80 transformation buffer (U.S. Patent 6,960,464) which is easier to make than the original Hanhan's buffer. On another note if you only care about transforming plasmids in and not other cloning procedures I would suggest Chung's DMSO method as I found it to be fastest (no cell washes involved). Find references and buffer below:
CCMB80 buffer:
http://cshprotocols.cshlp.org/content/2018/3/pdb.rec102707.short
Hanhan's paper:
Article Studies on transformation of Escherichia coli with plasmids
Chung's DMSO method:
https://benchling.com/protocols/PqDdikG7/tss-competent-e-coli-preparation
For electro competent cell preparation, you need just autoclaved milli-Q water. It can be stored for several months in 20% glycerol at -80 degree. But, you need to have electro cuvette and instrument.
DEar Saurav
commonly i'm using a protocool based on a first incubation with
100 mM MgCl2 - 30' in ice
and a second with
100 mM CaCl2, 15 % glicerol
which allow to reach competences of about 0,5-1*10^7cfu/ug for cells suitable for cloning (derived for K-12) as Top10, DH5alpha, DH10B and MACH1 or about 1*10^6 for BL21 (derived from E.coli B)
As Pierre Suggests to reach high competences (>10^8) you need to prepare elettrocompetent cells and perform elettroporation.
good luck
Manuele
Hi,
I would like to suggest you to go for CaCl2 method which works well for transformation. Additionally, give a try in transformation a freshly prepared competent cells with new vial.
Hope this helps.
Thank you all of you for the valuable suggestions. I would like to try Hanhan's method for my experiment.
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