Restriction Digestion

Experimental Objectives

To demonstrate the validity of Transcriptic’s Autoprotocol-implemented Restriction Digestion protocol. 

Methodology

A plasmid of 5712 bp was digested twice in succession using the Restriction Digestion Protocol. The plasmid contained unique restriction enzyme sites for BamH1 (base-pair 1355) and Nco1 (base-pair 2177). A total reaction volume of 45 µl was created using 2 µl of BamH1 and 4.5 µl of CutSmart bufer (both New England Biolabs), 10 µl DNA (at a concentration of 1 µg/µl) with 28.5 µl of distilled water. Reactions were performed for 30 minutes at 37°C. As part of the Restriction Digestion Protocol, 5 µl of total reaction volume was analyzed by gel electrophoresis, with a negative-control (enzyme free) reaction generated and analyzed in unison.

A second digestion, using the product mixture of the first reaction as DNA substrate was then generated using the Restriction Digestion Protocol. Two microliters of Nco1 (New England Biolabs) was combined with 3 µl of CutSmart bufer and 15 µl of the primary reaction mixture with 10 µl distilled water. Reactions were performed for 30 minutes at 37°C. As part of the Restriction Digestion Protocol, 5 µl of total reaction volume was analyzed by gel electrophoresis, with a negative-control (enzyme free) reaction generated and analyzed in unison.

Results

The Restriction Digestion Protocol was used to successfully perform sequential digestion of plasmid DNA. DNA fragments consistent with the anticipated sizes of 4890 and 822 bp were identified by gel electrophoresis, with comparative controls successfully generated.


VALIDATION DATE:  September 2015