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Invitrogen™ Subcloning Efficiency™ DH5α Competent Cells
Description
Includes
Subcloning Efficiency DH5α Competent Cells: 4 vials, 500μL each (total of 2mL), pUC19 DNA (100pg/μL): 1 vial, 20μL
Subcloning Efficiency DH5α Competent Cells are a versatile, chemically competent strain for cloning that provides a transformation efficiency of >1 x 106 cfu/μg plasmid DNA. Subcloning Efficiency DH5α Competent Cells are an economical solution for routine subcloning procedures or any general application where DNA for transformation is not limiting.
Features of Subcloning Efficiency DH5α Competent Cells
• Designed for general, low throughput, everyday use
• Standard laboratory strain useful for general cloning applications
Ideal for routine subcloning procedures
Subcloning Efficiency DH5α Competent Cells are recommended for routine subcloning of genes into plasmid vectors. 500 μL of chemically competent cells are pre-aliquoted in tubes for ten reactions using 50 μL. These cells yield >1 x 106 transformants/μg control DNA per 50 μL reaction. Subcloning Efficiency DH5α Competent Cells are not suitable for the generation of cDNA libraries.
Flexible cloning capabilities
Benefits of Subcloning Efficiency DH5α Competent Cells include:
• lacZΔM15 for blue/white color screening of colonies on plates containing X-gal or Bluo-gal
• recA1 that ensures increased insert stability and prevents unwanted recombination
• endA1 that improves the yield and quality of plasmid DNA prepared from minipreps
• Supports replication of M13mp vectors but not plaque formation
Genotype
F- Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 λ-
Find the strain and format that you need
• We offer other DH strains in chemically competent and electrocompetent cell formats to meet your specific needs.
• Strains are available for challenging cloning applications such as library preparation or cloning unstable DNA.
• SOC medium (Cat. No. 15544034) for E. coli recovery after transformation is available, as well as other ready-to-use bacterial growth formats.
Order Info
Shipping Condition: Dry Ice
Specifications
Specifications
| Product Type | Chemically Competent Cells |
| Contains F' Episome | No |
| Improves Plasmid Quality | Yes (endA1) |
| Cloning Methylated DNA | No |
| Transformation Efficiency Level | Subcloning Efficiency (106 to 107 cfu/μg) |
| Content And Storage | • Subcloning Efficiency DH5α Competent Cells (4 x 500 μL) Store Competent Cells at –80°C. • pUC19 DNA (20 μL at 100 pg/μL) Store pUC19 DNA at –20°C. |
| Antibiotic Resistance Bacterial | No |
| Cloning Unstable DNA | Not suitable for cloning unstable DNA |
| Blue/White Screening | Yes (lacZΔM15) |
| High-throughput Compatibility | Low |
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Frequently Asked Questions (FAQs)
If the insert is potentially toxic to the host cells, here are some suggestions that you can try:
- After transforming TOP10 or DH5? cells, incubate at 25-30°C instead of 37°C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
- Try using TOP10F' cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
- Try using Stbl2 cells for the transformation.
For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.
The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.
Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.
Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.
There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:
Subcloning Efficiency cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA
Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.
Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.
For Research Use Only. Not for use in diagnostic procedures.