New England Biolabs, C2988J, NEB® 5-alpha Competent E. coli (Subcloning Efficiency)

Having trouble opening your tubes? Related Categories Cloning Competent Cell Strains Applications Transformation Specification Antibiotic for Plasmid Selection Antibiotics for Plasmid Selection Working Concentration Ampicillin 100 µg/ml Carbenicillin 100 µg/ml Chloramphenicol 33 µg/ml Kanamycin 30 µg/ml Streptomycin 25 µg/ml Tetracycline 15 µg/ml Shipping Notes Ships on dry ice FAQ Q: Can LB medium be used instead of SOC in the outgrowth step (C2988)? A: NEB SOC outgrowth medium is made from animal-free peptone and delivers higher transformation efficiency than LB medium. All NEB competent cells (except Subcloning Efficiency NEB 5-alpha Competent E.coli [NEB#C2988J, NEB 10-beta competent E. coli [NEB#C3019][NEB#C3020K] and NEB Stable Competent E. coli [NEB#C3040]) contain SOC outgrowth medium in the kits. SOC outgrowth medium can also be purchased separately (NEB #B9020S)). Q: Can NEB 5-alpha competent E.coli (Subcloning Efficiency) (NEB #C2988J) be used for large fragment cloning? A: The transformation efficiency for C2988J is >1 X 10E6 cfu/μg pUC19, which is 1,000 fold less than other NEB high efficiency competent cells (1 X 10E9). If 50 μl of C2988J competent cells are transformed with 100 pg of pUC19 (2 μl of 50 pg/μl pUC19 control DNA [provided in NEB high efficiency competent cells]), 950 μl of LB should be added for outgrowth (LB only provides 28% transformation efficiency compared to SOC for this strain). If 100 μl is then spread onto a plate, only 2-3 colonies will be evident after overnight growth. We only recommend using subcloning efficiency competent cells (NEB #C2988J) for plasmid transformation or routine subcloning, such as inserting a 1kb fragment into a 2kb vector. A 0.8kb insert has successfully been cloned into pUC19 (2.7kb) with a transformation efficiency of 4.2 X 10E4 cfu/μg pUC19 vector. Q: What is the difference between NEB #C2988J and NEB #C2987H? A: They are the same NEB 5-alpha cells just provided in different transformation efficiency and different formats. C2987H is NEB 5-alpha competent E.coli with a high efficiency of 1-3X10E9 cfu/μg pUC19 DNA. C2988J is NEB 5-alpha competent E.coli with a subcloning efficiency of >1X10E6 cfu/μg pUC19 DNA. C2987H is packaged with 20 single-use transformation tubes containing 50 μl of competent cells each. Plasmid or ligation product can be added directly into the transformation tubes for convenience. C2988J is packaged with 6 tubes containing 400 μl of competent cells each. The tubes must be thawed on ice and 50 μl of cells should be pipetted into your own transformation tubes prior to transformation. Each tube contains enough cells for 8 transformations with the benefit of reducing the cost of each transformation. Refreezing the competent cells after thawing is not recommended since it will significantly reduce transformation efficiencies. Q: What is the optimal heat shock time for this strain (NEB #C2988J)? A: Heat shock at 42ºC for 30-40 seconds results in the highest transformation efficiency for NEB 5-alpha competent E.coli (Subcloning Efficiency) (NEB #C2988J). Expect approximately 40% loss in transformation efficiency when heat shocking for 80 seconds (see Figure on the main product page). Q: How long should I incubate cells on ice after DNA has been added (NEB #C2988J)? A: Incubating DNA with NEB 5-alpha competent cells (Subcloning Efficiency) on ice for 30 minutes is recommended. Expect approximately 30% loss in transformation efficiency when incubating for 10 minutes. (see Figure on the main product page). Q: How should I calculate the transformation efficiency of NEB 5-alpha Competent E. coli (Subcloning Efficiency)? A: Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 µg of plasmid into a given volume of competent cells. The term is somewhat misleading in that 1 µg of plasmid is rarely actually transformed. Instead efficiency is routinely calculated by transforming 100 pg-1 ng of highly purified supercoiled plasmid under ideal conditions. If you plan to calculate efficiency to compare cells or ligations, keep in mind the many variables which affect this metric. Transformation efficiency (TE) equation: TE = Colonies/µg/Dilution Colonies = the number of colonies counted on the plate µg = the amount of DNA transformed expressed in µg Dilution = the total dilution of the DNA before plating TE calculation example: Transform 1 µl (1 µg) of 1 mg/ml pUC19 DNA into 50 µl of cells, outgrow by adding 950 µl of SOC and dilute 10 µl up to 1 ml in SOC before plating 100 µl. If you count 250 colonies on the plate, the TE is: Colonies = 250 µg DNA = 1.0 Dilution = 10/1000 x 100/1000 = 0.001 TE = 250/1/0.001 = 2.5 x 105 cfu/µg Q: What are the solutions/recipes that I need for transforming NEB 5-alpha Competent E. coli (Subcloning Efficiency)? A: SOB: 2% Vegetable peptone (or Tryptone) 0.5% Yeast Extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 SOC: SOB + 20 mM Glucose LB agar: 1% Tryptone 0.5% Yeast Extract 0.17 M NaCl 1.5% Agar Blue/White Screening: X-gal 80 µg/ml IPTG* 0.3 mM *Omit IPTG for potentially toxic genes Q: What are the strain properties of NEB 5-alpha Competent E. coli (Subcloning Efficiency)? A: The properties of this strain that contribute to its usefulness as a cloning strain are described below. The genotypes underlying these properties appear in parentheses. Blue/White Screening: (Φ80 Δ(lacZ)M15 makes the omega-fragment of β-galactosidase (β-gal); (argF-lacZ) deletes the β-gal gene on the chromosome. pUC19 and similar plasmids code for the α-peptide of β-gal (lacZ). The α-peptide can combine with the omega-fragment of β-gal that is carried on f80 (α-complementation). When β-gal is reconstituted in this manner it can cleave 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal) and results in blue colonies on an X-gal plate. Inserts cloned into the plasmid polylinker disrupt the α-peptide gene and the colonies are white. Recombination Deficient: (recA1) E. coli has a repair system that will recombine homologous sequences. Genomic clones often have duplicated regions, and RecA mediated rearrangements can be problematic, particularly when regions of homology are longer than 50 bp. Strains which have the RecA function deleted tend to grow more slowly than recA+ strains. Endonuclease I Deficient: (endA1) The periplasmic space of wild type E. coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations. Restriction Deficient: (hsdR17) Wild type E. coli K12 strains carry a restriction endonuclease which cleaves DNA with sites (AAC(N6)GTGC and GCAC(N6)GTT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The hsdR mutation eliminates this endonuclease activity. However, this strain has functional methyl restriction systems and may not be suitable for direct cloning of eukaryotic DNA. T1 Phage Resistant: (fhuA2) T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell.DH5α™ is a trademark of Invitrogen Corporation. Q: Can I store competent cells at -20°C instead of -80°C? A: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency (TE). When tested on NEB 5-alpha Competent E.coli (NEB #C2987H), cells lost 94.5% of TE after only 24 hours of storage at -20°C. Cells lost 98.9% of TE after 2 days, and 99.6% of TE after one week of storage at -20°C. Q: Which kind of transformation tubes should be used? A: Compared to 2.0 ml tube provided with NEB single-use format competent cells, the 1.5 ml Eppendorf tube we tested worked just fine. Q: What volume of DNA can be added into competent cells? A: The volume of DNA to be added into competent cells does affect transformation efficiency. 1-5 µl of DNA (plasmid or ligation product) is recommended for 50 µl of competent cells. In 50 µl of competent cells, transformation efficiency drops to 52% when the DNA volume is increased to 10 µl (from 2 µl). Transformation efficiency drops to 18% when the DNA volume is increased to 20 µl (from 2 µl). Transformation efficiency drops to 5.2% when the DNA volume is increased to 50 µl (from 2 µl). Q: What is the shelf life for this strain (NEB #C2988J)? A: The expiration date is one year from the assay date provided with the product. Q: Are NEB's competent cells compatible with the “Mix & Go" protocol? A: There is a “ Mix and Go" protocol that provides a quick way to transform your cells by simply adding plasmid to cells and plating. No heat shock step is required. NEB has tested our competent cells in this protocol against another company's “Mix & Go” product. We have observed both will produce similar numbers of colonies; however, the NEB colonies are larger in size using the same incubation period. Q: How should fragments be prepared for assembly using NEBuilder HiFi? A: Fragments can be prepared by the following methods: PCR-generated fragments can be cleaned-up by using Monarch PCR column or Exo-CIP Rapid PCR Cleanup Kit if amplicon purity is greater than 95%. If plasmid DNA was used as template during PCR, it can be removed by DpnI treatment if necessary. If multi-bands are observed, we recommend optimizing the PCR. If this is not possible gel purification is recommended. Gel extraction can introduce guanidine thiocynate (from the dissolving buffer) that can reduce the efficiency of the assembly reaction. To minimize this contamination, trim the gel slice so that a smaller amount of gel dissolving buffer can be used. Restriction enzyme digestion of a plasmid can be performed followed by heat-inactivation or column purification. Commercially ordered fragments can be re-suspended in nuclease-free water or TE buffer and directly used in the assembly reaction.