Performed a restriction digestion on the mini prep DNA from 20120323 using NcoI (NEB). Digestion was set up as follows:
DNA (652.8ng) – 24uL
Buffer #3 – 2.8uL
H2O – 0.7uL
NcoI – 0.5uL
Reaction was incubated @ 37C for 2hrs and then heat-inactivated for 10mins @ 65C. Plasmid linearization was verified via 0.8% agarose TBE gel. 2uL of undigested plasmid DNA and 2uL of NcoI digested plasmid were each run on the gel.
Lane 1 – HyperLadder I (Bioline)
Lane 2 – Undigested mini prep plasmid DNA (2uL, ~54ng)
Lane 3 – NcoI digestion (2uL)
The undigested plasmid runs between 2500bp and 3000bp. The digested plasmid runs between 4000bp and 5000bp. Although I don’t know the size of the insert, this size for the linearized plasmid seems correct, since the empty vector (assumed to be pCR2.1) is 3900bp.
Things look good. Will proceed to quantifying the linearized plasmid and then proceed with making and evaluating a new standard curve for qPCR with the WSN1 primer/probe set.
20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.