Performed restriction digest using NcoI (NEB) to linearize plasmid for use as qPCR standard curve. Reactions were run for 1hr @ 37C and then heat inactivated @ 65C for 20mins.
Each reaction contained:
pCR2.1/Phage ORF25 (~1ug) – 17uL
10x Buffer 3 – 5uL
NcoI – 1uL
H2O – 27uL
After inactivation, 5uL from the reaction was run on a 1% TBE gel to confirm digestion. 5uL of undigested plasmid was run along side the digest.
Gel Loading Guide:
Lane 1 – Hyperladder I (Bioline)
Lane 2 – pCR2.1/ORF25 (undigested)
Lane 3 – pCR2.1/ORF25 (NcoI)
Vector size (bp): 3929
Insert size (bp): 483
Total size (bp): 4412
The linearized plasmid (which contains a single NcoI recognition site) runs between the 5000 and 4000bp standards, which is what we expect. Will quantify and generate dilution series for use as a qPCR standard curve.