Plasmid Isolation – Clam RLO 16s, EHR, & EUB clones

Prepared 1x LB + 50μg/mL ampicillin. Aliquoted 5mL of LB-Amp50 liquid media to 18 15mL conical tubes. Used the restreaked plates created 20150207 and used sterile pipette tips to select each of the six positive colonies from each cloning reaction and inoculate 5mL of LB-Amp50 liquid media. Tubes were incubated O/N @ 37C on a rocker.

All cultures grew. Three milliliters from each culture were used to isolate plasmid DNA using the QIAprep Spin Mini Kit (Qiagen). Samples were eluted with 50μL Buffer EB.

Frozen bacterial stocks were made from each of the six clones, using 500μL of each bacterial culture + 500μL of sterile, 50% glycerol in 2mL screw cap tubes.

Bacterial stocks and plasmid preps were labelled in the following fashion:

  • pCR2.1/Clam RLO 16s C1 – C6
  • pCR2.1/Clam RLO EHR C1 – C6
  • pCR2.1/Clam RLO EUB C1 – C6

where “C#” indicates the clone number. The bacterial stocks were stored @ -80C in the following box “Clones Box 2″:


Plasmid preps were quantified on the Roberts Lab NanoDrop1000.


Spreadsheet: 20150305_ClamRLO_miniprep_ODs

After speaking with Carolyn, she decided she wanted to sequence one clone from each group. Submitted ~500ng of clone #1 (C1) from each group to GENEWIZ for Sanger sequencing (Order #: 10-290123409). Each clone was sequenced from each direction with M13F (-21) and M13R primers for a total of 6 sequencing reactions:

1       SW01    16s_01-M13F(-21)
2       SW02    16s_02-M13R
3       SW03    EHR_01-M13F(-21)
4       SW04    EHR_02-M13R
5       SW05    EUB_01-M13F(-21)
6       SW06    EUB_02-M13R

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.


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.

Mini Prep – Bacterial Cultures from 20120409 & 20120410

Isolated plasmid DNA from cultures inoculated on 20120409 (p16RK3-C) & 20120410 (p16RK3-A). These cultures finally produced bacteria. Slow growth was likely due to anaerobic conditions (sealed 15mL conical tubes) and slow agitation (on a rocker instead of a shaker). Isolated plasmid DNA using Qiagen Mini Prep Spin Kit according to manufacturer’s protocol. Eluted plasmid DNA with 30uL of Buffer EB and quantified on Roberts Lab NanoDrop1000.


p16RK3-A = 134.2ng/uL

p16RK3-C = 36.4ng/uL

Mini Prep – RLP Clone 9

Performed mini prep using the Qiagen Mini Prep Spin Kit according to protocol on a 5mL O/N liquid culture (1x LB + 100ug/mL Amp) of RLP Clone 9 (from DATE). Sample was eluted with 30uL of Buffer EB and quantified using the Roberts Lab NanoDrop1000.