Sep 26, 2024
Bacteria Transformation
- 1Washington University
Protocol Citation: Carolina Lopez 2024. Bacteria Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn3kpgk5/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: May 28, 2024
Last Modified: September 26, 2024
Protocol Integer ID: 100763
Abstract
General protocol for bacteria transformation
Materials
Reagents:
- Competent E. coli cells (NEB, C3040H)
- LB plates with necessary antibiotic
- 50% Glycerol
Bacteria Transformation:
Bacteria Transformation:
1h 24m
1h 24m
1. Pipette 2 µL of product to 50 µL of E. coli competent cells and pipette slowly up and down 5 times to mix. For plasmids, use 2 µL ul of 0.005 µg/µL plasmid solution. For ligation reactions, DNA concentrations will vary so use 2 µL ul of reaction.
2. Incubate for 00:20:00 on ice.
3. Heat shock bacteria in 42 °C waterbath for 00:01:00 .
4. Incubate on Ice for 00:03:00 .
5. Add 100 µL of SOC media and shake in warm room for 01:00:00 .
6. Pipette 100 µL of bacteria onto LB-Antibiotic Plate and spread cells out with plate spreader to get individual colonies.
7. Incubate overnight in 37 °C warm room.
8. Pick colonies for miniprep growth and sequencing.
Glycerol Bacteria Stocks:
Glycerol Bacteria Stocks:
1. In screw top 2 mL tube. Mix 500 µL of Bacteria Solution (Either from Miniprep or Maxiprep before centrifugation) and 500 µL of 50% (v/v) Glycerol Solution.
2. Store at -80 °C .
3. To pull out bacteria, use bacteria loop to scrap some of still frozen glycerol stock and streak bacteria onto LB-Antibiotic plate. Then pick colonies for growth.