Transformation competent

Transform competent ceils with linearized cDNA... 

Transform competent coli host with recombinant vector and select for recombinants by antibiotic resistance appropriate for the plasmid. 

Transform competent coli (with a competency greater than 10 c.f.u./pig DNA) with 1 xl of the annealing reaction. Select for recombinants by plating on LB agar... 

The transformed competent cells are poured onto an agar plate and distributed by swirling. 

The plate coated with the transformed competent cells is incubated in an inverted position at 37°C for 12-16 h. 

Transformation competent E coli HB101 cells These can be prepared as described in ref 9, or can be obtained from commercial suppliers... 

Transform 10 pL each of the ligated DNAs into transformation-competent E coli HB101 cells. 

Transform competent E. coli according to the supplier s manual and cultivate in LB media (plates or liquid cultures, depending on the selection/screening approach see section 2.4, note 5). 

Seal in a capillary and incubate at 14°C for 6h. Use mixture to transform competent cells (e.g. JM101) and plate out (see Section 4.3.11. below). Count the number of plaques for each ligase concentration and choose the optimum concentration. NOTE Blunt-end ligation requires more ligase than sticky-end ligation of Accf-cleaved vector (2-base overlap, Fig. 4.5.) which requires more than ligation of EcoRI, Pstl or Bam Hi-cleaved vector (4-base overlap). 

DH5a transformation competent Escherichia coli (Gibco-BRL). 

Use 10 pL of the ligation reactions to transform competent E.coli using the methods described in Subheading 3.6.6. Store the remaining 10 pL of ligation mixture at -20°C in case the transformation needs to be repeated. 

Hartley et al. (1986) devised a nonradioactive probe, called probe-vector, which can, after hybridization, transform competent E. coli cells and, depending on the transformation efficiency, detect as little as 0.1 pg of target nucleic acid. The probe-vector molecules are linear, partially si DNA prepared by hybridizing individually prepared DNA strands. The ds region of the probe-vector encodes a phenotypic marker and origin of replication. The two terminal si... 

The epimerizalion of the 7a-hydroxyl group can occur either by intra- or interspecies mechanisms [16]. However, it is difficult to quantitatively assess the degree of 7-hydroxy epimerization in vivo because this transformation competes with the irreversible 7-dehydroxylation of bile acids (Section VI). 7a-HSDH activity has been reported in several genera of intestinal bacteria however, the most complete characterization of this enzyme has been carried out with the enzyme isolated from Escherichia coli [37] and Bacteroides sp. [29,38,39] (Table 2). Both enzymes used both free and conjugated bile acids as substrates, showed alkaline pH optima and lower values for dihydroxy than for trihydroxy bile acids. However, cell extracts prepared from Bacteriodes sp. contained both NAD- and NADP-depen-dent 7 -HSDH activities whereas, extracts from E. coli contained only an NAD-de-pendent enzyme activity. Additional studies showed that the two 7a-HSDH activities detected in Bacteriodes sp. differed in molecular weight, differential heat inactivation and Mn " requirement, suggesting the presence of two distinct enzymes [29]. 

Cloning and sequencing of amplicons. The amplified products were cloned into the pGEM-T vector (Promega) and used to transform competent E. coli DH5a. Transformed E. coli DH5 was then plated onto Luria-Bertani agar with 50 pg/mL... 

Once the plasmid has been created using conventional molecular cloning methods (Green Sambrook, 2012), it can be amplified by transforming competent Escherichia coli. 

Transform competent E. coli BL21 (DE3) cells with the pET28a-TPSTl expression plasmid and grow transformed E. coli at 37 °C in LB medium supplemented with 50 mg/ml of kanamycin. 

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