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Transformation of plasmid DNA to competent E. coli cells
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.
·If a lower efficiency is sufficient, we use heat shock transformation and chemically competent cells.
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. 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.
·The transformation of a vector for multiplication should also be done in a recA- strain, such as DH5a, NovaBlue or XL1-Blue.
·The transformation of a vector for protein expression should be done in the appropriate expression host (see table).
Expression vector appropriate host strain
pBAD vectors Top10, LMG194
pET vectors BL21 (DE3)
pGEX vectors BL21
pMal vectors BL21, TB1
pProEx vectors BL21, DH10B
pQE vectors M15 or M15 [pREP4]
pRSET vectors BL21 (DE3) pLysS
pTrcHis vectors BL21
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.
·If a lower efficiency is sufficient, we use heat shock transformation and chemically competent cells.
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. 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.
·The transformation of a vector for multiplication should also be done in a recA- strain, such as DH5a, NovaBlue or XL1-Blue.
·The transformation of a vector for protein expression should be done in the appropriate expression host (see table).
Expression vector appropriate host strain
pBAD vectors Top10, LMG194
pET vectors BL21 (DE3)
pGEX vectors BL21
pMal vectors BL21, TB1
pProEx vectors BL21, DH10B
pQE vectors M15 or M15 [pREP4]
pRSET vectors BL21 (DE3) pLysS
pTrcHis vectors BL21
