Frequently Asked Questions - EB5Alpha Chemically Competent Cells

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(cfu on control plate) / (ng of supercoiled vector) x (103ng / µg) x (final dilution) = cfu / µg DNA

(Note: cfu = colony forming units)

Transformation efficiency usually refers to the number of cells that are transformed by 1µg DNA, usually a supercoiled plasmid.

These are cells that have been physically manipulated to increase their transformation efficiency

Transformation is the process by which naked DNA is introduced into cells. It also can happen naturally at a low frequency

Yes, you can add 250µl SOC directly to the 1.5ml transformation tube and shake it at 300 rpm for 1 hour without altering the transformation efficiency.

The following table shows recovery done with 1ml SOC in 14ml round bottom culture tubes. The percentages are relative to 60 minutes of recovery:

Minutes
Transformation efficiency using pET41 plating in LB/Kan
Transformation efficiency using pUC19 plating in LB/amp
 
 
 
60
100
100
45
76
87
30
19
60
15
2
63
0
0
59

Standard SOC medium can be prepared as follows:

Ingredients Final Concentration
Yeast Extract 5g/l
Tryptone 20g/l
Sodium Chloride 10mM
Potassium Chloride 2.5mM
   
Autoclase, then add:  
Glucose 20mM
Magnesium Chloride 10mM
Magnesium Sulfate 10mM

Glucose and magnesium present in SOC seem to improve transformation efficiency. Our transformation efficiencies are calculated using a standard 60-minute incubation with SOC after the transformation.

After bacteria have been transformed with an antibiotic-resistant plasmid, recovery in medium lacking the antibiotic allows for synthesis of the antibiotic resistance prior to plating them in the presence of antibiotic. The importance of recovery depends on the antibiotic used. For example, recovery is essential for kanamycin-resistant plasmids; whereas, ampicillin-resistant plasmid recovery improves the transformation efficiency 2-5 fold.

EB5Alpha Competent Cells is a K12 derivative with similar growth. Generally, overnight incubation of plated transformed cells in LB/antibiotic at 37ËšC results in medium size colonies (about 1mm in diameter).

We calculate the transformation efficiency of our competent cells with 10pg supercoiled pUC19 and expect a transformation efficiency of at least 2x109 colonies/µg pUC19 supercoiled DNA.

No, transformation efficiency, although proportional to the number of clones generated, is only one of many factors that will determine the number of clones after ligation of library construction. Other factors include: amount of vector used, quality of DNA, size of DNA, method of generating DNA (i.e. PCR, miniprep, etc.), restriction enzyme used (blunt or cohesive ends), systems used to generate clones (i.e. recombination or conventional ligation) and others. EB5Alpha Competent Cells have been thoroughly controlled for quality and are guaranteed to exceed transformation efficiencies of 109 colonies/µg pUC19 supercoiled DNA.

Competent cell stability in terms of transformation efficiency is guaranteed by Edge BioSystems for at least 3 months, when properly stored at -70°C. Upon receipt of your cell shipment, cells should immediately be placed at -70°C to ensure optimal activity.

We compared transformation efficiencies of supercoiled pUC19 vs. ligated pUC19 after single and double cut. We also tested if heat activation of the ligase improved transformation efficiency. The results are shown below. Please note that in order to make the experiments comparable, transformations were not done according to our standard protocols. Transformation efficiencies with standard protocols exceed 109cfu/µg pUC19.

Material (pUC19) Transformation Efficiency % Efficiency Relative to Supercoiled pUC19
Supercoiled pUC19 5 x 108 100
Single cut pUC19, heat inactivated ligation 6.2 x 108 100
Single cut pUC19 5.4 x 108 100
Double cut pUC19, heat inactivated ligation 5.4 x 107 10.8
Double cut pUC19 3.9 x 107 7.8

The more DNA used, the more efficient the ligation will be. The ratio of vector to inserted DNA can be critical for obtaining high-efficiency ligations. A molar excess of insert to vector may yield higher efficiency ligations when subcloning inserts into plasmid vectors. However, an equal or greater ratio of vector to insert may be preferred when performing library construction into plasmid vectors.

EB5Alpha Competent Cells are best suited for applications that include a need for increased plasmid yield and quality, ultra efficient blue/white screening for cloning and generation of cDNA libraries. Although EB5Alpha cells can be used for protein expression, Edge BioSystems proudly offers its Ultra BL21 (DE3) and Ultra BL21 (DE3) pLysS Competent Cells, which have proven to be very successful in protein expression applications.

This kit is intended for internal research use only by the purchaser. It is not to be used for human diagnostic purposes. In addition, "research use only" means that this kit and all of its contents are excluded from resale, repackaging or use for the making or selling of any commercial product or service without the written approval from Edge BioSystems.

EB5Alpha Competent Cells offers distinct advantages in particular applications. Blue/white screening and high transformation efficiencies make them ideal for all cloning applications and generation of libraries. They are especially suitable for high-throughput cloning applications. Their genetic background provide excellent plasmid yield and quality with peace of mind for phage T1 and T5 resistance.

Edge BioSystems guarantees >109 colonies / µg pUC19. As with any other competent cell, the transformation efficiency will decrease with larger plasmids or ligated DNA.

These cells are ideal for most cloning needs and for the generation of plasmid-based libraries. Their high efficiency makes them the cells of choice for cloning experiments using limiting amounts of DNA in either a high-throughput or single use format. Blue/white color selection facilitates screening of positive clones.

EB5Alpha:   F-Φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-,mk+) phoA supE44 thi-1 gyrA relA1 tonA

Genotype Advantage
lacZΔM15 Allows blue/white screening through β-galactosidase complementation.
recA1 Eliminates general DNA recombination.
endA1 Improves yield and quality of DNA in plasmid preparations.
hsdR17(rk-,mk+) Represents restriction negative and modification positive phenotype and allows cloning of DNA without cleavage by endogenous restriction endonucleases.
relA1 Permits RNA synthesis in the absence of protein synthesis.
tonA Confers resistance to bacteriophages T1 and T5.

Different strains are clearly labeled as such. While we do not label the aluminum seal affixed to the plate product, there are other distinguishing labels that make identification easy. Labeling the aluminum seal would prevent the piercing characteristic.