Aug 20, 2020
Bacterial Transformation - Mix & Go Competent Cells - CHEM 584
- 1Brigham Young University
Protocol Citation: Ken Christensen 2020. Bacterial Transformation - Mix & Go Competent Cells - CHEM 584 . protocols.io https://dx.doi.org/10.17504/protocols.io.bj2tkqen
Manuscript citation:
Adapted from https://files.zymoresearch.com/protocols/_t3001_t3002_mix_go_e._coli_transformation_kit_buffer_set.pdf
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: In development
We are still developing and optimizing this protocol
Created: August 20, 2020
Last Modified: August 20, 2020
Protocol Integer ID: 40755
Safety warnings
You should follow standard BSL-1 procedures when using this protocol.
Before start
Get an aliquot of the Mix & Go competent cells from the -80 Freezer (ask a TA to get them for you)
Prewarm culture plates in a 37 °C incubator for 00:30:00 .
Add 1 µL to 5 µL plasmid DNA to a 50 µL aliqanuot of thawed competent cells (Mix & Go) on ice, mix gently for a few seconds (try to keep the added volume of DNA less than 5% of the total). Place tube on ice for 00:05:00 minutes.
Note
When selecting with Kanamycin, Tetracycline, etc., an outgrowth performed in SOC medium is required for efficient transformation. After the transformation mixture has incubated on ice for at least 5 min, add 4 volumes of SOC (e.g., 400 μl of SOC to 100 μl of the transformation mixture) and incubate for 1 hour at 37°C with gentle shaking at 200-300 rpm.
Spread 50 µL to 100 µL of the mixture onto a pre-warmed (37°C) LB plate containing Ampicillin (or other selection antibiotic).
Note
Using chilled LB plates will decrease the transformation efficiency.
Incubate overnight at 37 °C in the bacterial incubator.
Note
You can also incubate on the bench or in a drawer at room temperature for a longer time if this is convenient for scheduling. For example, leaving a plate on the bench for 2-3 days at room temperature usually generates nice sized colonies and you avoid having to come into the lab the next day (e.g., over the weekend).