Technical Bulletin E. coli Competent Cells INSTRUCTIONS FOR USE OF PRODUCTS L1OO1, L1O11, L1191, L12O1, L2OO1 AND L2O11. o E. coli Competent Cells All technical literature is available on the Internet at: www.promega.com/tbs/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on use of this system. E-mail: techserv@promega.com 1. Description..........................................................................................................1 2. Product Components and Storage Conditions............................................2 3. Standard Transformation Protocol.................................................................3 4. Calculation of Transformation Efficiency....................................................5 5. Composition of Buffers and Solutions.........................................................6 6. References...........................................................................................................7 7. Related Products................................................................................................7 1. Description The E. coli Competent Cells are prepared according to a modified procedure of Hanahan (1). The competent cells can be used for many standard molecular biology applications. Competent cells of strains HB101 and JM109 are available for convenient transformation in two efficiencies: High Efficiency at greater than 108cfu/ng and Subcloning Efficiency at greater than 107cfu/ng. JM109 cells (2) are an ideal host for many molecular biology applications. HB101 cells (3) are useful for cloning in vectors that do not require a-complementation for blue/white screening. The BMH 71-18 mutS strain is suitable for use in in vitro mutagenesis procedures where a repair (-) strain is required. BL21(DE3)pLysS cells(a) can be used with protein expression vectors that are under the control of the T7 promoter, such as pET vectors. This strain is lysogenic for lambda-DE3 (4) , which contains the T7 bacteriophage gene 1, encoding T7 RNA polymerase (5) under the control of the lacUV5 promoter. BL21(DE3)pLysS also contains the pLysS plasmid, which carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved following induction with IPTG. For genotypic information on the E. coli Competent Cells, see Table 1. Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Printed in USA. Part# TB095 Revised 3/09 Page 1 o Promega Table 1. Genotypes of £. coli Competent Cells Offered by Promega. Strain_Genotype_ BL21(DE3)pLysS BMH 71-18 mutS laqIqZD M15] HB101 JM109 F-, ompT, hsdSB (rB-, mB-), dcm, gal, 1DE3), pLysS, Cm1 thi, supE, D (lac-proAB), [mutS::Tn10], [FC, proAB, F-, thi-1, hsdS20 (rB-, mB-), supE44, recA13, ara-14, leuB6, pro A2, lacYl, galK2, rpsL20 (strr), xyl-5, mtl-1 endA1, recA1, gyrA96, thi, hsdR17 (rk-, mk+), relA1, supE44, D (lac-proAB), [FC, traD36, proAB, laqIqZD M15] 2. Product Components and Storage Conditions Product Size Cat.# JM109 Competent Cells, 1ml L2001 >108cfu/ng* (5 x 200ul) JM109 Competent Cells, 1ml L1001 >107cfu/ng (5 x 200ul) HB101 Competent Cells, 1ml L2011 >108cfu/ng (5 x 200ul) HB101 Competent Cells, 1ml L1011 >107cfu/ng (5 x 200ul) BL21(DE3)pLysS Competent Cells, 1ml L1191 >106cfu/ng (5 x 200ul) BMH 71-18 mutS Competent Cells, 1ml L1201 >107cfu/ng (5 x 200ul) * For Laboratory Use Storage Conditions: Always store Competent Cells at -70°C. Thaw on ice when ready for use. Do not refreeze thawed, unused aliquots. All cells are supplied in 200(i aliquots and provided with 3ng of Competent Cells Control DNA for use as a positive control. Typically, 100(i of Competent Cells are required for a standard transformation. Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Part# TB095 Printed in USA. Page 2 Revised 3/09 o Promega 3. Standard Transformation Protocol Materials to Be Supplied by the User (Solution compositions are provided in Section 5.) • LB or SOC medium • LB plates with antibiotic • 17 x 100mm polypropylene culture tubes, sterile (e.g., Falcon™ 2059) • IPTG (Cat.# V3955; optional, see Note 4) • X-Gal (Cat.# V3941; optional, see Note 4) 1. Chill sterile 17 x 100mm polypropylene culture tubes on ice, one per transformation (e.g., Falcon™ 2059). Use of a standard microcentrifuge tube reduces the transformation efficiency by approximately 50% due to inefficient heat-shock treatment of the cells. 2. Remove frozen Competent Cells from -70°C, and place on ice for 5 minutes or until just thawed. Once the cells have thawed, pipet quickly or use chilled (4°C) pipette tips to prevent the cells from warming above 4°C. 3. Gently mix the thawed Competent Cells by flicking the tube, and transfer 100[d to each chilled culture tube. 4. Add 1-50ng of DNA (in a volume not greater than 10[d) per 100[d of Competent Cells. Move the pipette tip through the cells while dispensing. Quickly flick the tube several times. Note: To determine the transformation efficiency, we recommend using 1jd (0.1ng) of Competent Cells Control DNA at this step. 5. Immediately return the tubes to ice for 10 minutes. 6. Heat-shock the cells for 45-50 seconds in a water bath at exactly 42°C. Do not shake. 7. Immediately place the tubes on ice for 2 minutes. 8. Add 900[d of cold (4°C) SOC medium to each transformation reaction, and incubate for 60 minutes at 37°C with shaking (approximately 225rpm). Note: Use high-quality deionized water (e.g., Milli-Q® or NANOpure®) for SOC medium (see recipe in Section 5). If LB or other medium is used, transformation efficiencies will be reduced. 9. For each transformation reaction, we recommend diluting the cells 1:10 and 1:100 and plating 100[d of undiluted cells and 1:10 and 1:100 dilutions on antibiotic plates (see Notes 1-3). Incubate the plates at 37°C for 12-14 hours. Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Printed in USA. Part# TB095 Revised 3/09 Page 3 o Promega 3. Standard Transformation Protocol (continued) Notes: 1. For transformations using the Competent Cells Control DNA, we recommend diluting the cells 1:10, then plating 100^1 on LB/ampicillin plates. 2. Do not dilute BL21(DE3)pLysS Competent Cells; spread 100[d of these cells directly onto antibiotic plates. 3. If desired, pellet the cells by centrifugation at 1,000 * g for 10 minutes, then resuspend in 200[d of SOC or LB medium and plate (see note at Step 8). 4. Blue/white screening can be used with a variety of vectors in conjunction with JM109 Competent Cells. To use blue/white color screening for recombinants, plate the transformed cells on LB plates containing 100ng/ml ampicillin, 0.5mM IPTG (Cat.# V3955) and 40ng/ml X-Gal (Cat.# V3941). Incubate overnight at 37°C. An alternative to preparing plates containing X-Gal and IPTG is to spread 20[U of 50mg/ml X-Gal and 100jd of 0.1M IPTG onto LB ampicillin plates, and allow these components to absorb for 30 minutes at 37°C prior to plating cells. Note: HB101 and BL21(DE3)pLysS Competent Cells cannot be used for blue/white color screening. 5. Solutions and media containing tetracycline must be stored protected from light to maintain potency. Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Part# TB095 Printed in USA. Page 4 Revised 3/09 o Promega 4. Calculation of Transformation Efficiency (Colony Forming Units [cfu]) Transformation efficiency is defined as the number of colony forming units (cfu) produced by lug of Competent Cells Control DNA (supercoiled plasmid DNA) and is measured by performing a control transformation reaction using a known quantity of DNA, typically O.lng, then calculating the number of cfu formed per microgram DNA. Note: The Competent Cells Control DNA (pGEM®-3Z Vector) is supplied at a concentration of O.lng/ul in TE buffer. Equation for Transformation Efficiency (cfu/ug) cfu on control plate x l x 103ng ng of Competent Cells Control DNA plated ug Example: After adding 9OOul of SOC medium to lOOul of competent cells that have been transformed with O.lng Competent Cells Control DNA, transfer lOOul (equivalent to O.OlngDNA) to 9OOul of SOC medium and plate lOOul (equivalent to O.OOlng DNA). If lOO colonies are observed on the plate, the transformation efficiency is: lOOcfu x l x lO3ng , x 1O8 f , -r-r-r-- X -Q = l X lO8cfu/ug O.OOlng ug Note: Transformation with ligated plasmid DNA will produce fewer colonies than transformation with supercoiled plasmid DNA. Promega Corporation • 28OO Woods Hollow Road • Madison, WI 537ll-5399 USA Toll Free in USA 8OO-356-9526 • Phone 6O8-274-433O • Fax 6O8-277-25l6 • www.promega.com Printed in USA. Part# TBO95 Revised 3/O9 Page 5 o Promega 5. Composition of Buffers and Solutions glucose, 2M 180.16g glucose Add distilled water to 500ml, filter-sterilize through a 0.2[im filter unit and store in aliquots at -20°C. Stable for 1 year. IPTG stock solution, 0.1M 1.2g IPTG (Cat.# V3955) Add water to 50ml final volume. Filter-sterilize through a 0.2[im filter unit, and store at 4°C. LB medium with ampicillin 10g/L Bacto®-tryptone 5g/L Bacto®-yeast extract 5g/L NaCl Adjust the pH to 7.5 with NaOH. Autoclave to sterilize. Allow the autoclaved medium to cool to 55°C, and add ampicillin (final concentration 100ng/ml). For LB plates, include 15g agar prior to autoclaving. X-Gal Available from Promega (Cat.# V3941) at a concentration of 50mg/ml in dimethylformamide. Mg2+ stock solution, 2M 101.5g MgCl2« 6H2O 123.3g MgSO4 • 7H2O Add distilled water to 500ml, and filter-sterilize through a 0.2^m filter unit. Note: Filter-sterilizing units should be prerinsed with distilled water before use to remove any toxic material. SOC medium 2.0g Bacto®-tryptone 0.5g Bacto®-yeast extract 1ml 1M NaCl 0.25ml 1M KCl 1ml Mg2+ stock (1M MgCl2 • 6H2O, 1M MgSO4 • 7H2O), filter-sterilized 1ml 2M glucose, filter-sterilized Bring to 100ml with distilled water. Add Bacto®-tryptone, Bacto®-yeast extract, NaCl and KCl to 97ml distilled water. Stir to dissolve. Autoclave and cool to room temperature. Add 2M Mg2+ stock and 2M glucose stock, each to a final concentration of 20mM. Filter the complete medium through a 0.2[im filter unit. The pH should be 7.0. Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Part# TB095 Printed in USA. Page 6 Revised 3/09 o Promega 6. References 1 Hanahan, D. (1985) In: DNA Cloning, Vol. 1, Glover, D., ed., IRL Press, Ltd., 109. 2. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-19. 3. Lacks, S. and Greenberg, B. (1977) Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114, 153-68. 4. Studier, F.W. and Moffatt, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-30. 5. Davanloo, P. et al. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035-9. 7. Related Products Competent Cells Product Size Cat.# Single Step (KRX) Competent Cells 5 x 200uJ 20 x 50uJ L3001 L3002 Bacterial Strains (not competent cells) Product Size Cat.# Bacterial Strain JM109, Glycerol Stock 500jü P9751 Bacterial Strain JM109(DE3), Glycerol Stock 500jü P9801 Bacterial Strain BL21(DE3)pLysS, Glycerol Stock 500jü P9811 Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Printed in USA. Part# TB095 Revised 3/09 Page 7 o Promega (a)Usage Restrictions for the T7 Expression System The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy and is the subject of patents assigned to Brookhaven Science Associates, LLC (BSA). This technology, including bacteria, phage and plasmids that carry the gene for T7 RNA polymerase, is to be used for academic or nonprofit laboratory or licensed commercial research purposes only. By accepting or using the T7 expression technology you agree to be bound by the following conditions set forth by BSA. The initial purchaser may refuse to accept the conditions of this notice by returning this product and the enclosed materials to Promega unused. Academic and NonProfit Laboratories No materials that contain the cloned gene for T7 RNA polymerase may be distributed further to third parties outside of your laboratory unless the recipient receives a copy of this assurance notice and agrees to be bound by its terms. This limitation applies to Bacterial Strains JM109(DE3), BL21(DE3)pLysS and KRX and to any derivatives thereof. Commercial Laboratories A license is required for any commercial use of the T7 expression system, including use of the T7 system for research purposes or for production purposes by any commercial entity. Information about commercial licenses may be obtained from the Licensing Office, Brookhaven National Laboratory, Upton, NY 11973, Telephone: 631-344-7134, FAX: 631-344-3729. © 1990-2009 Promega Corporation. All Rights Reserved. pGEM is a registered trademark of Promega Corporation. Bacto is a registered trademark of Difco Laboratories, Detroit, Michigan. Falcon is a trademark of Becton, Dickinson and Company. Milli-Q is a registered trademark of Millipore Corporation. NANOpure is a registered trademark of Barnstead/Thermolyne Corporation. Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information. All prices and specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. Promega Corporation • 2800 Woods Hollow Road • Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 • Phone 608-274-4330 • Fax 608-277-2516 • www.promega.com Part# TB095 Printed in USA. Page 8 Revised 3/09