Mar 05, 2024
Transforming pJC8 into HB101
- Bonnie Evans1,2
- 1MRC Laboratory of Medical Sciences;
- 2Imperial College London
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
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
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
Before starting
Prepare tetracycline LB agar plates
Protocol
NAME
Making tetracycline LB agar platesCREATED 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
Transformation
Isolate pJC8 cosmid from E. coli DH5α
Protocol
NAME
Qiagen QIAprep Spin Miniprep for cosmids from metagenomic libraryCREATED 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.