1. You have a lot of competent cells. How do I know which ones to use?
Here is a table of the cells and their intended use. Most of Lucigen’s competent cell strains come in both chemically and electrocompetent versions. Table 1. Lucigen Competent Cells
|Strain Name ||Application |
|E.cloni® 10G and 10GF’ ||General Cloning and library construction |
|TransorMax™ EC100™ ||General Cloning and library construction |
|E.cloni 5-alpha ||General Cloning and library construction |
|TG1 ||Phage Display |
|SS320 ||Phage Display |
|ER2738 ||Phage Display |
|Endura™ ||Difficult Cloning (Low Recombination/unstable inserts) |
|TransforMax EPI300™ ||Difficult Cloning (Copy number control/toxic proteins) |
|TransforMax EPI400™ ||Difficult Cloning (Copy number control/toxic proteins) |
|BAC-Optimized Replicator™ V2.0 ||Large/Difficult Cloning (Large inserts) |
|10G BAC-Optimized ||Large/Difficult Cloning (Large inserts) |
|BigEasy®-TSA ||Large/Difficult Cloning (Large inserts/Copy number control) |
|CJ236 ||Kunkel site-directed mutagenesis |
|OverExpress™ C41(DE3) ||Protein Expression (toxic proteins) |
|OverExpress C43(DE3) ||Protein Expression (toxic proteins) |
|E.cloni EXPRESS BL21(DE3) ||Protein Expression (overexpression) |
|Hi-Control™ BL21(DE3) ||Protein Expression (tight expression) |
|Hi-Control 10G ||Protein Expression (tight expression) |
|ClearColi® BL21(DE3) ||Protein Expression (No Endotoxin) |
2. What’s a SOLO, or a DUO?
Lucigen comp cells are supplied in SOLO and DUO formats. SOLO’s provide enoµgh cells for 1 transformation per tube, and DUOS provide enoµgh cells for 2 transformations per tube.
3. Can I thaw a DUO and re-freeze the rest of the cells that I don’t use?
No, any unused thawed cells should be discarded. Re-freezing cells for later use will result in a loss of transformation efficiency.
4. How should I store the cells?
In order to maintain the highest levels of transformation efficiency and product performance, it is critical the cells remain frozen at all times. Upon receipt of cells:
- Open shipping container and remove cooler.
- Open cooler and pour off dry ice pellets into a safe container.
- IMMEDIATELY remove Lucigen competent cells and place in a -80°C freezer.
5. Can I store competent cells at -20°C instead of -80°C?
No, competent cells rapidly lose their ability to take up DNA at -20°C.
6. What is the shelf life for my competent cells?
The expiration date is one year from the date of shipment. Cells are likely to be useable past this date when maintained at -80°C, however, Lucigen cannot guarantee optimal performance past the expiration date.
7. Do you test each lot of cells for transformation efficiency?
Yes. Every lot of cells is tested to ensure the cells meet our guaranteed transformation efficiencies. We use supercoiled pUC19 plasmid DNA to determine efficiency, unless otherwise noted in the product manual.
Each lot of custom cells is also tested. Note that for customer-provided cell strains, we cannot guarantee a specific transformation efficiency.
8. Do you perform other QC tests, in addition to determining transformation efficiency?
Yes, each lot of cells (for catalog kits
and all custom orders
) is tested for resistance to a panel of antibiotics by plating mock-transformed cells on antibiotic plates. Common antibiotic panels include: kanamycin, carbenicillin (ampicillin), streptomycin, tetracycline, gentamycin and chloramphenicol. Lucigen cells must pass strict QC specifications for growth/no growth on appropriate antibiotics.
9. What volume of DNA can be added to competent cells?
No more than 10% of the volume of the cells. Transformation efficiency drops rapidly past this point.
10. How long should I incubate cells on ice after DNA is added?
For chemically competent cells, incubation time on ice is strain-dependent. General ranges are from 5-30 minutes before heat shock and 2 minutes after heat shock. For electrocompetent cells, DNA should be added to cells and electroporated immediately in a pre-chilled electroporation cuvette.
11. What type of tube should I use for my transformation?
When using chemically competent cells, we sµggest transferring cells to a pre-chilled sterile 15 mL culture tube (17mm x 100mm) before adding DNA. For electrocompetent cells, transfer immediately after electroporation to a pre-chilled 15 mL culture tube, then add Recovery Medium.
12. What is the optimal heat shock time for a transformation?
Optimal time varies between strains, but general guidelines are between 30 and 45 seconds. Follow the recommendations in the product manual.
13. What is the antibiotic selection on the positive control plasmid?
Cells are provided with supercoiled pUC19 DNA. Plate transformants on plates containing 100 µg/mL ampicillin or carbenicillin.
14. Within the phage display strains, what is the difference between an amber suppressor and non-amber suppressor strain?
Amber suppressor strains contain suppressor mutations that read throµgh amber stop codons (TAG/UAG). Instead of translation termination, another amino acid is supplied and protein synthesis continues. Two of Lucigen’s phage display cell lines (TG1 and ER2738) are amber suppressor strains.
15. Can I use E. cloni® 10G cells for protein expression?
16. Should I use electrocompetent or chemically competent cells to make my library?
Lucigen recommends using electrocompetent cells for library generation, because the transformation efficiency for electrocompetent cells is higher. Higher transformation efficiency preserves library diversity.
17. Does Lucigen have strains that can be used with Gateway® cloning?
Lucigen strains such as E. cloni® 10G, Endura™ and TransforMax™ EPI300™ and EC100™ can be used for transforming Gateway cloning reactions and selection of recombinant plasmids. However, these Lucigen strains should not be used for propagating or preparing the empty Gateway donor or destination plasmids, which contain the ccdB gene and must be grown in strains that are resistant to the effects of the ccdB gene.
Strains that contain the F’ plasmid (such as E. cloni 10G F’) contain the ccdA gene and are therefore resistant to the ccdB gene. Therefore these strains could be used for propagating and preparing ccdB-containing backbones, but should not be used for transforming Gateway cloning reactions and selecting recombinant plasmids.
18. Which competent cells can be used with Gibson Assembly®?
Most E. coli strains are compatible with Gibson Assembly. SGI-DNA, the inventors of Gibson Assembly cloning, endorse two Lucigen strains for use with their Gibson Assembly kits. The high efficiencies of these cell strains have been found to increase colony counts after transformation of Gibson Assembly reactions:
E. cloni 10G Chemically Competent Cells
TransforMax™ EPI300™ Electrocompetent Cells
19. What competent cells would you recommend for difficult cloning?
Lucigen has competent cells that solve multiple cloning problems. Endura™ cells are excellent for maintaining sequences with inverted repeats or other sequences prone to recombination, such as those found in viral vector backbones. The CopyCutter™ EPI400™ cells are used to maintain toxic proteins. Lucigen also has multiple strains that can be used to clone large DNA fragments for libraries, such as BAC-Optimized Replicator™ cells. Contact our Technical Support team for recommendations for your specific project.
20. How do I calculate transformation efficiency (TE)?
TE = (# colonies) / (µg DNA transformed) * (final transformation volume) / (volume plated) * dilution factor = cfu / µg
For example, if you transformed 1 ng of plasmid DNA into cells, diluted the transformation to a final volume of 1000 µL with Recovery Medium, then plated 25 µL of a 1:100 dilution of that transformation, and the next day you counted 150 colonies on your plate, your calculation would look like this:
# of colonies: 150
µg DNA transformed: 0.001
Final tansformation volume = 1000 µL
Volume plated = 25 µL
Dilution factor = 1 µL into 100 µL, 100-fold (factor of 100)
TE = (150 / 0.001) * (1000 / 25) * 100 = 6 x 108 cfu / µg
21. Why do I get a lower transformation efficiency than Lucigen advertises?
As common practice, Lucigen uses purified supercoiled pUC19 DNA to determine transformation efficiency. Many customers use ligation mixtures to transform cells, which do not transform as efficiently as supercoiled plasmid. Different plasmid backbones may not transform as well, and some inserts may be toxic. DNA purity can also affect efficiency. Also, make sure that the competent cells have been consistently stored at the recommended temperatures. Storage at warmer temperatures can cause transformation efficiency to drop precipitously.
22. Can I heat shock electrocompetent cells and get transformants?
No. Electrocompetent cells are manufactured and formulated specifically for electroporation, and will not take up DNA after heat shock.
23. There are transformation protocols for chemically competent cells that do not include a heat shock. Do I need to heat shock my competent cells?
We have not tested alternative transformation protocols extensively. You can avoid the heat shock step, but it is highly likely that your transformation efficiencies will decrease.
24. Does plasmid size affect transformation efficiency?
Generally, the larger the plasmid, the less efficiently it is transformed.
25. What are the efficiencies of your cells in a 96-well format?
E. cloni 10G Chemically Competent cells have a transformation efficiency of are 1 x 108 cfu/µg in 96-well plates (Cat #60096-1 and 60096-4). We routinely deliver our other competent cell strains in 96-well plates on a custom basis, with customer-specified dispense volumes. Transformation efficiency can vary based on cell strain and the volume of cells dispensed into the well.
26. Why are my colonies so small?
Check the agar media. Lucigen recommends using LB-Lennox (low salt), rather than LB-Miller (high salt), for plating most of our cell lines. We’ve found colonies grow larger after overnight incubation at 37°C on LB-Lennox.
Note that for ClearColi® cell lines, LB-Miller (high-salt) media is required.
27. Why does Lucigen recommend carbenicillin instead of ampicillin?
Resistance to both ampicillin and carbenicillin is conferred by the b-lactamase enzyme. Ampicillin is easily broken down, resulting in small, background colonies known as “satellite colonies”. Carbenicillin is more stable.