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.

Plasmid Isolation – Withering Syndrome Phage pCR2.1/ORF20 and pCR2.1/ORF25

From cloning on 20140908, inoculated 5mL of 1x LB + 100ug/mL ampicillin with pCR2.1/ORF20 clone # 7 and pCR2.1/ORF25 clone #1. Incubated O/N @ 37C on rocker. Isolated plasmids with QIAprep Mini Kit (Qiagen) according to the manufacturer’s spin protocol, using ~3mL of culture. Eluted plasmid DNA with 100uL of EB Buffer.

Frozen bacterial stocks of each were made. 1mL of each culture was mixed with 1mL of 50% glycerol (sterile) and stored @ -80C

NOTE: Ended up diluting the pCR2.1/ORF25 by adding an additional 100uL of EB Buffer because it seemed too concentrated for proper quantification using the plate reader.

Spec’d plasmid DNA using Teacan plate reader and Pico green dye, according to lab protocol.

Results:

Spec’ing via the Teacan plate reader is not working (see separate entry regarding this).

Spec’d using NanoDrop1000:

Plasmids look great; excellent yields and quality. Will prepare some for linearization for use as standard curves and will use some for making a probe for in-situ hybridization.