Competent cells with very high transformation efficiencies are used for DNA library construction (such as cDNA, gDNA, phage display, and CRISPR and gene libraries) to promote high coverage of unique constructs. Electroporation is a preferred transformation method to create DNA fragment and gene libraries because it provides the highest transformation efficiency. Competent cells for heat-shock transformation are also available.
How critical is transformation efficiency for getting good DNA library representation
E. coli strains for plasmid library construction
These competent cells have mutated mcrA, mcrBC, mrr, and/or hsdRMS restriction systems so they are efficient at cloning methylated DNA, which is important for construction of highly representative eukaryotic genomic libraries.
The following Invitrogen competent cells feature:
- Blue/white colony selection
- High transformation efficiency
Electrocompetent bacteria for library preparation
| Product |  |  |  |  |
|---|---|---|---|---|
| ElectroMAX DH10B Competent Cells | ElectroMAX Stbl4 Competent Cells | ElectroMAX DH10B T1 Phage-Resistant Competent Cells | MegaX DH10B T1R Electrocompetent Cells | |
| Recommended for | gDNA and plasmid rescue | Inverted and direct repeats, CRISPR libraries | gDNA and plasmid rescue (phage resistance) | Large DNA inserts, highest efficiency |
| Transformation Efficiency | >1 x 1010 cfu/µg | >5 x 109 cfu/µg | >1 x 1010 cfu/µg | >3 x 1010 cfu/µg (highest) |
| Compatible plasmid size | >20 kb | >100 kb | >20 kb | >100 kb |
| T1, T5, and φ80 phage resistance (tonA (fhuA)) | – | – | ||
| Generating ssDNA (Contains F´ episome) | – | – | – | |
| Cloning unstable inserts | – | – | – | |
| M13mp18 RF DNA included | – | – | – | – |
| Format | 5 x 100 µL/tube | 5 x 100 µL/tube | 5 x 100 µL/tube | 5 x 100 µL/tube |
Chemically competent bacteria for library preparation
| Product |  |  |
|---|---|---|
| MAX Efficiency DH5αF´IQ Competent Cells | OmniMAX 2 T1R chemically competent E. coli | |
| Recommended for | Phage display, toxic gene cloning | Gateway and TOPO cloning reactions |
| Transformation Efficiency | >1 x 108 cfu/µg | >5 x 109 cfu/µg |
| Compatible plasmid size | >20 kb | >20 kb |
| T1, T5, and φ80 phage resistance (tonA (fhuA)) | – | |
| Generating ssDNA (Contains F´ episome) | ||
| Cloning unstable inserts | – | – |
| M13mp18 RF DNA included | – | |
| Format | 5 x 200 µL/tube | 21 x 50 uL/tube |
Electrocompetent bacteria for library preparation
| Product | | | | |
|---|---|---|---|---|
| ElectroMAX DH10B Competent Cells | ElectroMAX Stbl4 Competent Cells | ElectroMAX DH10B T1 Phage-Resistant Competent Cells | MegaX DH10B T1R Electrocompetent Cells | |
| Recommended for | gDNA and plasmid rescue | Inverted and direct repeats, CRISPR libraries | gDNA and plasmid rescue (phage resistance) | Large DNA inserts, highest efficiency |
| Transformation Efficiency | >1 x 1010 cfu/µg | >5 x 109 cfu/µg | >1 x 1010 cfu/µg | >3 x 1010 cfu/µg (highest) |
| Compatible plasmid size | >20 kb | >100 kb | >20 kb | >100 kb |
| T1, T5, and φ80 phage resistance (tonA (fhuA)) | – | – | ||
| Generating ssDNA (Contains F´ episome) | – | – | – | |
| Cloning unstable inserts | – | – | – | |
| M13mp18 RF DNA included | – | – | – | – |
| Format | 5 x 100 µL/tube | 5 x 100 µL/tube | 5 x 100 µL/tube | 5 x 100 µL/tube |
Chemically competent bacteria for library preparation
| Product | | |
|---|---|---|
| MAX Efficiency DH5αF´IQ Competent Cells | OmniMAX 2 T1R chemically competent E. coli | |
| Recommended for | Phage display, toxic gene cloning | Gateway and TOPO cloning reactions |
| Transformation Efficiency | >1 x 108 cfu/µg | >5 x 109 cfu/µg |
| Compatible plasmid size | >20 kb | >20 kb |
| T1, T5, and φ80 phage resistance (tonA (fhuA)) | – | |
| Generating ssDNA (Contains F´ episome) | ||
| Cloning unstable inserts | – | – |
| M13mp18 RF DNA included | – | |
| Format | 5 x 200 µL/tube | 21 x 50 uL/tube |
Phage display library construction
Phage display libraries are used to discover antibodies [1] and have been used to identify human antibodies against SARS-CoV-2 [2]. In addition to high transformation efficiency competent cells, phage display protocols require a M13KO7 Helper Phage that can be acquired separately.
CRISPR library construction
CRISPR screening workflows often require construction of sgRNA libraries. Stbl4 competent cells have been used to successfully build these RNA libraries [3].
Preparing cDNA and genomic DNA libraries
Thermo Fisher Scientific provides many reagents and resources for bacterial library construction. cDNA can be constructed using the Invitrogen Second Strand cDNA Synthesis Kit or other cDNA library construction kits.
For genomic DNA library preparation, use Invitrogen genomic DNA extraction kits to consistently achieve excellent results.
Tips for transforming bacteria for construction of DNA libraries
- Ledsgaard L., Kilstrup M., Karatt-Vellatt A., et al. (2018) Basics of Antibody Phage Display Technology. Toxins (Basel) 236. doi:10.3390/toxins10060236
- Wu Y., Li C., Xia S., et al. (2020) Identification of Human Single-Domain Antibodies against SARS-CoV-2. Cell Host & Microbe 891. doi:10.1016/j.chom.2020.04.023
- Read A., Gao S., Batchelor E., et al. (2017) Flexible CRISPR library construction using parallel oligonucleotide retrieval. Nucleic Acids Res 45. doi:10.1093/nar/gkx181