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.
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