Restriction Digestion – Withering Syndrome Phage ORF25 Plasmid from 20140926

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

Total: 50uL

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.

Results:

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.

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