Klonierung und DNA-Marker

Subcloning is a basic procedure in molecular biology that is used to move inserts from one vector to another to gain desired functionality and to characterize a DNA sequence of interest. 

One method to accomplish the transfer of a DNA insert uses restriction enzymes to digest both the fragment and the target vector, which is typically a plasmid. Confirmation of successful digestion is accomplished by comparing the expected size of the digested samples with DNA marker fragments. This is done by running samples on agarose gels to separate DNA fragments by size.  T4 DNA Ligase is then used to “paste” the desired fragment into the digested plasmid.

The next step is transformation. Transformation of bacteria with plasmids is important because bacteria are used as the means for both storing and replicating plasmids. E. coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Such cells are said to be competent. Selection for cells that have been transformed successfully is done by antibiotic selection for the plasmid of interest.

There are many options to confirm if the transfer was successful including PCR, digestion with restriction enzymes or sequencing. Once confirmed, the recombinant vector can be used for a variety of applications such as protein expression or RNA transcription.