Selectable markers

E.coli K12 TGI were grown to log phase (up to OD6oo=0.20-0.30) in Luria-Bertani (LB) broth, washed and ultimately concentrated 25 times in ice-cold 100 mM of CaCb. DNA was extracted from agarose gel after electrophoresis, added to 200 ml of competent cell and incubated at 0°C for 15 min. The cell-DNA complex was transferred to 42°C for exactly 90 s and was rapidly chilled in ice. Then 1000 ml LB-broth was added and the cells were incubated at 37°C for 60 min. 100 ml cells was spread on LB-agar with and without selective marker ampicillin (50 mg/ml), to obtain the number of transformants and viable cells respectively. Plates were incubated at 37°C for 18-24 h. 

In Vivo Enrichment of Gene-Protected Cells by Coexpression of a Selectable Marker... 

DNA construct will often contain an effect gene and a selectable marker gene (such as antibiotic or herbicide resistance), both of which are bracketed by promoter and terminator sequences. A plasmid vector carries this cassette of genetic information into the plant genome by one of the above methods. 

Figure 19 Sample gel of the results of a PCR. Lane 1 is a 100-bp molecular marker lanes 2-6 are samples. The presence of the top bands (the species-specific endogenous gene) demonstrates that the PCR amplification was successfiil. Lack of the middle band (the introduced effect gene) and the bottom band (the selectable marker gene) in lane 3 indicates that sample is negative for the effect gene. Presence of all three bands in the remaining lanes indicates the samples are positive for the effect gene...

The plant sample in lane 6 is also positive for the transgene of interest. Because the band for the effect gene (middle band) is typically fainter than the band for the selectable marker gene (bottom band), it appears that for lane 6, the PCR product amplitication for the effect gene is below the assay detection threshold. Because the selectable marker is clearly present and the PCR amplitication worked, lane 6 can be interpreted as a positive result for the transgene of interest. 

Flanking counter-selectable markers Eliminates vector backbone Reduces transformation efficiency 23,24... 

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