Aug 29, 2022
pCP20 transformation to remove Kanamycin cassette
- 1Imperial College London, MRC London Institute of Medical Sciences
- Saul Moore: This protocol was carried out by Cassandra Backes of the Host-Microbe Co-Metabolism laboratory
Protocol Citation: Saul Moore 2022. pCP20 transformation to remove Kanamycin cassette. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbydxovpk/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: August 28, 2022
Last Modified: August 29, 2022
Protocol Integer ID: 69281
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Abstract
Transformation with pCP20 to remove the Kanamycin cassette from Keio E. coli BW215113 single gene deletion mutants - Cassandra Backes (Host-Microbe Co-Metabolism Laboratory, MRC-LMS)
Materials
Making TSS broth(for final volume 10 mL): Make fresh!
·10% (w/v) Polyethylene Glycol (PEG) 3350* – on media shelf; add 1 g
·LB broth – add 9 mL
·50 mM Mg2+ (MgSO4 or MgCl2) – use 1M stock of MgSO4 used for NGM; add 500 uL
·Sterilise-filter into new tube
·5% (v/v) DMSO –chemical metal chest; add 500 uL
Making TSS enhanced buffer (for final volume of 10 mL):
·Add 8.2 mL of dH2O to 30 mL tube
·100mM KCl – (to prepare 1M stock: 1.491 g in 20 mL of H2O); add 1 mL
·30mM CaCl2- use 1M stock for NGM; add 0.3 mL
·50mM MgSO4 - use 1M stock for NGM; add 0.5 mL
·Vortex well and filter-sterilise into 2 mL tubes
·Keep in fridge
*There are other molecular weights of PEG, so look at label carefully and choose one with 3350 on it!
Make buffer and broth the day before and keep them in the fridge
Day before: Grow O/N cultures of the strains of interest. Can also prepare buffers (see materials).
Dilute overnight culture in LB broth at an OD = 0,2 and grow until OD595nm = 0.5 – 0.8 (mid-log phase).
UNTIL NOW, KEEP CULTURES ON ICE
Spin down culture(s): transfer first to 15 mL falcon, spin 4500 RPM, 10 min, 4°C
In the meantime, label 2 mL tubes with strain names and add 80uL TSS buffer and 1 - 5 uL of pDNA (50-75ng). vortex and keep on ice for 10 min.
Remove supernatant from 14 mL tubes and resuspend pellet in 1mL TSS broth(cold). Resuspend gently and keep on ice.
Then transfer cells to a 2 mL tube.
Add 200uL of bacterial culture in TSS broth to the 2 mL tubes containing pDNA in TSS buffer, Mix gently by pipetting.
Incubate for 20 min on ice.
Add 1mL LB.
Shake at 30oC for 1 hour 700 rpm.
Spin down (4500 RPM, 5 min), resuspend pellet in 300uL LB.
Spread 50 and 150ul on a plate and incubate at 30C in Chloramphenicol plates!
Keep remaining transformation mix at 4oC.
Check plates next day for single colonies, and confirm by PCR/Sequencing.