Tag Archives: plasmid isolation

Plasmid Isolation – RLOv pCR2.1 Clones

Clone #1 was selected from each of the screened clones.

A sterile pipette tip was used to inoculate 5mL of 1x LBAmp100 in a 15mL conical tube. The tubes were incubated O/N @ 37C on a rocker.

3mL of liquid culture was used as input for plasmid isolation with the QIAprep Spin Mini Kit (Qiagen) according to the manufacturer’s protocol.

1mL of each culture was combined with 1mL of 50% sterile glycerol (25% glycerol final concentration) and stored @ -80C with existing bacterial stocks.

Plasmid DNA was eluted with 50μL of Buffer EB and quantified on the Roberts Lab NanoDrop1000 (ThermoFisher) in order to have a rough idea of concentrations to submit for Sanger sequencing. A dye-based quantification will be performed after sequencing results are back in order to obtain a more accurate assessment for use in ISH and/or qPCR standard curve creation.

Results:

Quality (260/280 & 260/230 ratios) look great and yields are more than sufficient. Will prep samples for Sanger sequencing.

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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.

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