I have been trying to amplify NEB 5-alpha F'Iq Competent E. coli for my phage display library and therefore need a high transformation efficiency. The commercial product has a TE of 10^9, but I have tried the chemical transformation cells amplification protocol (using Inoue method) and also electrocompetent method (using ice cold 10% glycerol) and got only 10^5 TE resp. Please help me troubleshoot or suggest some ideas if anybody is familiar with the strain. Thank you.
NEB product no: C2992I
https://international.neb.com/products/c2992-neb-5-alpha-fiq-competent-e-coli-high-efficiency#Product%20Information
it is quite difficult, and almost an art, to get very high efficiency competent cells. You need to be very meticulous in every step of the procedure. For routine work we always used homemade cells and they are fine, but if really high efficiency is needed, we always purchased commercial competent cells. We were never able to get them as good as the "professionals" could do. Although it should be not that hard to get 10e6 or 10e7, we never got 10e9. This is true for really any standard cloning strain, nothing magical about the NEB strain.
Hi
I agree with the comments above that making high efficiency chemical competent cells is tricky, but can be achieved. However, the electrocompetent cells with high efficiency is more easy to achieve. The only factors need to be optimize is the initial number of cells and appropriate field strength to be used to get a balance of minimum possible cell killing with maximum possible transformation efficiency. Different E. coli strains show different field strength which is optimum for the maximum transformation efficiency which can be achieved. There are few old publications on this aspects also. Do it systematically, it should work
Surely Michael‘s answer is the general condition. I did try once to reach almost 10^9, but I never got that later. we question whether some batches of electrocompetent are superior and if lucky you get them and easily reach the required diversity, normally 10^7 per shot is the major case(Gibson assembled product). my suggestion is, if you want to repeatedly get such diversity, let different people in the lab prepare homemade electrocompetent, surely some guys would be able to prepare superior electrocompetent. it really require some luck~
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