Transforming pJC8 into HB101
Bonnie Evans
Abstract
Taken from Mix and Go E. coli Transformation Kit (see attachment).
Transforming E. coli HB101 with pJC8-empty cosmid to use as a control strain.
Attachments
Steps
Before starting
Prepare tetracycline LB agar plates
Warm plates in 37 °C incubator
Prepare SOC medium
Get SOB medium and 20 % glucose from media kitchen
Add 2 mL 20 % glucose to 100 ml SOB medium
Transformation
Isolate pJC8 cosmid from E. coli DH5α
Qiagen QIAprep Spin Miniprep for cosmids from metagenomic library
Thaw 100 uL aliquot of E. coli HB101 Mix & Go competent cells on ice
Add 5 uL pJC8 cosmid
Mix by tapping the tube and shaking downwards once
Place on ice for 5 minutes
Add 400 uL SOC medium
Incubate at 37 °C with shaking at 200 rpm for 1 hour
Pipette all (505 uL) of the transformation mix onto a tetracycline LB agar plate and spread using plating beads.
Dry under laminar flow for a few minutes
Incubate plate at 37 °C overnight
