Mar 05, 2024

Transforming pJC8 into HB101

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj3kkwlk5/v1
  • 1MRC Laboratory of Medical Sciences;
  • 2Imperial College London
Bonnie Evans
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvj3kkwlk5/v1
Protocol Citation: Bonnie Evans 2024. Transforming pJC8 into HB101. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3kkwlk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 05, 2024
Last Modified: March 05, 2024
Protocol  Integer ID: 96163
Keywords: pjc8 into hb101, transforming pjc8, pjc8, hb101, empty cosmid, control strain
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
Icon for _t3001_t3002_mix_go_e._coli_transformation_kit_buffer_set.pdf
_t3001_t3002_mix_go_...
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Materials
Mix and Go E. coli Transformation Kit (Zymo Research, T3001/T3002)

Contents of SOB medium:
20g Bacto Tryptone
5g Yeast Extract
0.58g Sodium Chloride
0.19g Potassium Chloride
1L sterile H20

Before starting
Prepare tetracycline LB agar plates
Protocol
NAME
Making tetracycline LB agar plates
CREATED BY
Bonnie Evans

Warm plates in 37 °C incubator

Note
Chilled plates will decrease Mix & Go cell transformation efficiency.

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
Protocol
NAME
Qiagen QIAprep Spin Miniprep for cosmids from metagenomic library
CREATED BY
Bonnie Evans

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

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

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

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