DNA effects on transformation efficiency and colony output The optimal amount of DNA to use in a transformation reaction is lower than commonly recognized. Using clean, supercoiled pUC19, the efficiency of transformation is highest in the 100 pg-1 ng range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 ng.
DNA effects on transformation efficiency and colony output
The optimal amount of DNA to use in a transformation reaction is lower than commonly recognized. Using clean, supercoiled pUC19, the efficiency of transformation is highest in the 100 pg-1 ng range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 ng.Growth curve of NEB turbo vs DH5α at 37°C in shaking flask
NEB Turbo and DH5α frozen stock (same cell density) were inoculated 1:100 into 80 ml of liquid medium (10g tryptone, 5g yeast extract, 10g NaCl, 1g MgCl2 and 1g Dextrose per liter, pH7.2) in 1 liter flask, respectively. Cells were grown at 37°C with shaking at 250 rpm. O.D.600 was measured every 30 minutes for up to 16 hours.Growth curve of NEB turbo vs DH5α at 37°C in fermentor NEB Turbo and DH5α cultures grown overnight were inoculated 1:50 into 1 liter of LB (10g tryptone, 5g yeast extract, 10g NaCl per liter, pH7.0) in 1 liter fermentor, respectively. Cells were grown at 37°C. O.D.600 was measured every 30 minutes for up to 9 hours.NEB turbo cell growth and DNA yield
pUC19 was transformed into NEB Turbo competent E.coli. Six colonies grown overnight (colony size was about 2.4 mm in diameter) were inoculated into 30 ml of LB/Amp, mixed thoroughly and 5 ml was dispensed into each 25 ml test tube (equal to 1 colony in 5 ml of LB/Amp). The cells were grown at 37°C with shaking at 250 rpm. One test tube was placed on ice after 3, 4, 5, 6, 7 and 16 hours of growth, respectively and the O.D.600 was measured. Mini-prep was performed by spinning down 1.5 ml of culture 3 times (total 4.5 ml) and finally eluting the DNA into 50 μl of EB buffer.Comparison of DNA yield after 4 hours of growth pUC19 was transformed into NEB Turbo competent E.coli, Invitrogen Mach1 and DH5α, respectively. One overnight grown colony (colony size for NEB Turbo was about 2.4 mm in diameter, 1.8 mm for Mach1 and 1.2 mm for DH5α) was inoculated into 5 ml of LB/Amp in a 25ml test tube and cells were grown at 37°C with shaking at 250 rpm. After 4 hours of growth, cells were measured O.D.600 and mini-prep was performed by spinning down 1.5 ml of culture 3 times (total 4.5 ml) and finally eluting the DNA into 50 μl of EB buffer. O.D.600 for NEB Turbo was 3.08, 2.36 for Mach1 and 0.75 for DH5α.Effect of heat shock time on NEB turbo competent E.coli transformation efficiency
50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except heat shock time varied from 0 to 80 seconds.
Effect of DNA incubation time on NEB turbo competent E.coli transformation efficiency
50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except DNA incubation time varied from 0 to 40 minutes.Effect of outgrowth medium on transformation efficiency 50 μl of NEB Turbo competent E.coli was transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol with the exception of varying the outgrowth medium. NEB SOC outgrowth medium delivers the highest transformation efficiency.
CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
