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.
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.