840:153g:Projects/project27/2012/10/16

Plasmid Backbone Isolation

• The pSB1K3 and J61002 plasmids were digested with Xba1 and Pst1 enzymes in order to remove inserted parts. pSB1K3 contained a 1022bp insert including parts R0010 (Lac1), B0034, E1010 (mRFP1), and B0015. J61002 contained a ~845bp insert including J23100, E1010 (mRFP1), and B0015. Digested plasmid DNA was run on a gel to separate plasmid backbone from insert DNA. Gel bands containing plasmid backbone were excised and stored for DNA recovery.

The three wells to the left of the ladder contained pSB1K3 samples, while the two wells to the left of the ladder contained J61002 samples. Two pSB1K3 samples and one J61002 sample showed DNA. The bands near the 1000bp marker (furthest downward on the gel) contain the plasmid inserts that were digested out. The bands between 2000 and 3000bp contained plasmid backbones and were separated from adjacent bands (undigested plasmid) and excised from the gel.