Excellent Cheap Way to Make Competent E.coli

June 25, 2014

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California Institute of Technolgy
Biology
Post-doctoral research scholar

Company:

Zymo Research

Product Name:

Mix & Go! E.coli Transformation Kit (up to 20 ml)

Catalog Number:

T3001

My research focuses on T cell immunotherapy using lentiviral vectors. For this, I need to keep cloning various inserts into lentiviral backbones as well as some TOPO cloning. I used this product because of how expensive commercially produced competent cells are.

Experimental Design and Results Summary

Application

Molecular Cloning

Starting Material

E coli (DH5a), Mix and Go competent cell kit

Protocol Overview

Grow DH5a E coli in 50 ml Zymo-Broth (supplied) for 16-18 hours at room temperature with shaking at 225 rpm. Keep the culture on ice for 10 minutes. After this, harvest the bacteria by centrifugation at 1600g for 5 min at 4 degC. Resuspend gently in 5 ml of 1X ice cold wash buffer. Centrifuge again at 1600g for 5 min at 4 degC. Resuspend in 5 ml of 1X competent buffer. Aliquot in 1.5ml tubes and freeze at -80 degC. For transformation: Thaw E coli on ice. Add 1-5 ul of DNA per 50 ul cells. Keep on ice for 30 seconds. Spread on a pre-warmed LB plate. Keep overnight at 37 degC.

Tips

COLD .. Do not ever take E coli out of ice/4 degC during the whole protocol. Grow E coli at room temperature for better efficiency.

Results Summary

Using this protocol, I get 0.5-2x10^7 CFU/microgram of pUC19 routinely. The great thing about this product is that it takes ~25 min to make one batch of DH5a. Just by resuspending in 5 ml competent cell buffer, you get 50x100 ul aliquots of competent Cells. Subcloning efficiency DH5a from Invitrogen will cost you $80 for 2 ml of cells. Those cells are about 1x10^6 CFU/microgram. The mix and go kit will cost you $104 for up to 20 ml of cells. That is a saving of ~$700.

Additional Notes

None

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Summary

The Good

Very cheap, very simple and robust kit

The Bad

Nothing.. Except that you have to spend ~25 minutes making cells

The Bottom Line

If you are willing to spend half an hour of your time to save $700 on competent cells, this is the kit for you! I highly recommend this kit for making competent cells.

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