Transforming pJC8 into HB101

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.

_t3001_t3002_mix_go_e._coli_transformation_kit_buffer_set.pdf

Prepare tetracycline LB agar plates

Making tetracycline LB agar plates

Warm plates in 37 °C incubator

Note

Chilled plates will decrease Mix & Go cell transformation efficiency.

Prepare SOC medium

3.1.

Get SOB medium and 20 % glucose from media kitchen

3.2.

Add 2 mL 20 % glucose to 100 ml SOB medium

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

10.

Incubate at 37 °C with shaking at 200 rpm for 1 hour

Note

An outgrowth in SOC medium is required for efficient transformation when selecting with tetracycline.

11.

Pipette all (505 uL) of the transformation mix onto a tetracycline LB agar plate and spread using plating beads.

12.

Dry under laminar flow for a few minutes

13.

Incubate plate at 37 °C overnight

Citation

If the transformation is successful, you will have individual colonies.