transform, Spread, pick

Overview

Bacterial transformation is the process by which exogenous DNA is introduced and incorporated into competent bacterial cells. Routinely used in molecular biology, this arduous and time-consuming workflow is essential for cloning as well as propagation and maintenance of plasmid DNA (double-stranded circular DNA). This protocol automates transformation, spread and pick on a Transcriptic Workcell; freeing researchers from redundant activities.  

This protocol takes up to 32 DNA aliquots of your choice and Zymo Mix & Go [1] (Zymo Research Corporation) chemically competent cells (Zymo 5α, Zymo 10B, or JM109) to generate individually picked colonies from streaked bacteria. Picked colonies are grown for 16 hours at 37°C to prepare for downstream applications such as plasmid preparation and/or Sanger Sequencing. Colony growth is monitored by an endpoint absorbance reading at 600nm. 

Methodology

50ul of chemically competent Zymo Mix & Go cells (Zymo 5α, Zymo 10B, or JM109) are provisioned and thawed for each transformation. Up to 32 DNA specified by the customer per run is added and mixed with the bacterial cells. Zymo Mix & Go cells do not require heat shock during transformation. 

The DNA-bacteria mixture is incubated at 4°C for 20 minutes. An optional SOC recovery step adds nutrient-rich SOC broth, and incubates and shakes the mixture at 37°C for 10 minutes. Bacteria is spread on 6-well agar plates containing selection media (Figure 1) and plates are incubated at 37°C for 16 hours. 

Following growth, the user-specified number of monoclonal colonies are picked into 96-well flat plates containing media with selection antibiotic. Pick plates are grown at 37°C for 16 hours and an endpoint absorbance reading is taken at 600nm to assess bacterial growth (Figure 2). Individually picked colonies are ready for your analysis and downstream applications such as Sanger Sequencing or plasmid preparation. 

Validation

To demonstrate the validity of this Transcriptic protocol, 10 constructs were assembled using the Kunkel Mutagenesis protocol and subsequently used for transformation, spread and pick. Zymo10B Mix & Go cells were transformed with each of these assembled constructs, spread on agar plates with selection media (Figure 1), and grown for 16 hours. 

Figure 1  Representative image of a 6-well agar plate spread with transformed bacteria and grown for 16 hours at 37°C. Each test assembly was chemically transformed and plated on 6- well agar plates containing selection media. The agar plate was imaged following 16 hours of growth at 37°C.

Figure 1  Representative image of a 6-well agar plate spread with transformed bacteria and grown for 16 hours at 37°C. Each test assembly was chemically transformed and plated on 6- well agar plates containing selection media. The agar plate was imaged following 16 hours of growth at 37°C.

To test pick, three colonies for each of the 10 assemblies as well as a negative control were picked, grown for 16 hours at 37°C, and growth was validated by an endpoint absorbance reading at 600nm (Figure 2). All picked colonies displayed absorbance readings >0.3 whereas colonies picked from the negative control had no growth (0.05). 

Figure 2  Representative absorbance data at 600nm for picked bacterial colonies following 16 hour outgrowth. For each assembly (1-10) along with a negative control, three colonies were picked into a 96-well flat plate containing selection media, and grown for 16 hours at 37°C. Bacterial growth was determined by an endpoint absorbance reading at 600nm. 

Figure 2  Representative absorbance data at 600nm for picked bacterial colonies following 16 hour outgrowth. For each assembly (1-10) along with a negative control, three colonies were picked into a 96-well flat plate containing selection media, and grown for 16 hours at 37°C. Bacterial growth was determined by an endpoint absorbance reading at 600nm. 

Conclusion

Transcriptic’s automated platform provides a robust and efficient method for executing bacterial transformation on demand. Zymo Mix & Go cells are chemically transformed with your DNA of choice. Following spread and growth, individually picked colonies are ready-to-go for plasmid preparation, Sanger Sequencing, and other downstream applications. 


[1]  Zymo Mix & Go chemically competent cells do not require heat shock during transformation.


VALIDATION DATE: November 2015
PROTOCOL EXECUTED: Transform, Spread, Pick