Transformation is the process of getting the recombinant vector from a reaction mixture or vector solution into E. coli cells. To enable the cells to take up circular vector DNA they have to be made competent. The method for the preparation of competent cells depends on the transformation method used and transformation efficiency required.
- For a high transformation efficiency, we use electroporation and electroporation-competent cells. Here is a protocol for preparing electrocompetent E. coli.
- If a lower efficiency is sufficient, we use heat shock transformation and chemically competent cells. Here is a protocol for preparing heat shock competent E. coli
The choice of the E. coli host strain depends on the goal of the transformation.
- The transformation of a ligation mix should be done in a recA- cloning strain, such as DH5a, NovaBlue or XL1-Blue. Always use a negative control with only vector DNA.
- Depending on the background of non-recombinants (from a ligation mix containing only digested vector), a number of transformants (3-12) should be picked and checked for the presence of the right insert by restriction analysis or colony PCR.
Note: We recommend colony PCR testing prior to DNA Minipreps as you can get the result for positive clones while the cultures inoculated with the same colonies are incubating. You prepare DNA only from
1-2 of the PCR-positive clones instead of preparing from all inoculated colonies and use restriction digestion for testing. It will save you time and money for restriction enzyme and Miniprep columns, additionally you can screen more colonies in parallel.
(Should your PCR not work for some reason, at least you still have your colonies growing, therefore you don't lose anything while waiting for them to grow.)
- The transformation of a vector for multiplication should also be done in a recA- strain, such as DH5, NovaBlue or XL1-Blue.
- The transformation of a vector for protein expression should be done in the appropriate expression host (see table below).
appropriate host strain
|pBAD vectors||Top10, >LMG194|
|pET vectors||BL21 (DE3)|
|pMal vectors||BL21, TB1|
|pProEx vectors||BL21, DH10B|
|pQE vectors||M15 or M15 [pREP4]|
|pRSET vectors||BL21 (DE3) pLysS|