One Shot TOP10 Chemically Competent E. coli

1 One Shot TOP10 Chemically Competent E. coliCatalog Number C4040-10 (10 reactions), C4040-03 (20 reactions), and C4040-06(40 reactions)Doc. Part No. Pub. No. MAN0001491 Rev. ! This product contains irritants and may be harmful if the Material Safety Data Sheet before handling. Safety Data Sheets(SDSs) are available from descriptionTOP10 E. coli are provided at a transformation efficiency of 1 109 cfu/ g supercoiledDNA and are ideal for high-efficiency cloning and plasmid propagation. They allowstable replication of high-copy number plasmids.

2 The genotype of TOP10 Cells issimilar to the DH10B and storageComponentQuantityStoragecondition sTOP10 Chemically Competent E. coli Cells21 50 LStore at 80 CpUC19 Control DNA (10 pg/ L)50 Medium6 mLPRODUCT INFORMATION SHEETFor Research Use Only. Not for use in diagnostic guidelinesFollow these guidelines when using Invitrogen One Shot TOP10 ChemicallyCompetent E. coli. Handle Competent cells gently as they are highly sensitive to changes in temperatureor mechanical lysis caused by pipetting. Thaw One Shot Competent cells on ice, andtransform cells immediately following thawing.

3 After adding DNA, mix by swirlingor tapping the tube gently. Do not mix cells by pipetting. One Shot TOP10 cells do not require IPTG to induce expression from the lacpromoter. If blue/white screening is required to select for transformants, make surethat selective plates contain 50 g/mL X you beginPerform the following before starting the transformation a water bath to 42 the vial of Medium (supplied with the kit) and LB Medium (ifneeded) to room X-gal onto LB agar plates containing antibiotic, if the selective plates in a 37 C incubator for 30 minutes (use 1 or 2 plates foreach transformation).

4 If you are including the pUC19 control, make sure that youhave one LB agar plate containing 100 g/mL transformation efficiencyUse the following formula to calculate the transformation efficiency as transformants(in cfu) per g of plasmid DNA. Remember that the total volume of the transformationmixture is 300 factor# of colonies10 pg pUC19 DNA106 pg g300 L total volume X L plated xxxTransformation efficiency (# transformants/ g DNA) = If transformation of 10 pg of pUC19 DNA yields 100 colonies when 30 L of a 1:10dilution is plated, then the transformation efficiency is:10 = 1 x 109 cfu/ g100 colonies10 pg DNA106 pg g300 L total volume 30 L plated xxx2 One Shot TOP10 Chemically Competent E.

5 Coli Product Information SheetTransform Competent cellsUse this procedure to transform One Shot TOP10 Chemically Competent E. coli. Werecommend including the pUC19 control plasmid DNA supplied with the kit(10 pg/ L in 5 mM Tris-HCl, mM EDTA, pH 8) in your transformation experimentto verify the efficiency of the Competent cells. Do not use these cells for : For a rapid transformation procedure, see Transform Competent cells (rapidtransformation procedure) on page , on ice, one vial of One Shot TOP10 Chemically Competent cells for 1 to 5 L of the DNA (10 pg to 100 ng) into a vial of One Shot cells and mixgently.

6 Do not mix by pipetting up and down. For the pUC19 control, add 10 pg(1 L) of DNA into a separate vial of One Shot cells and mix the vial(s) on ice for 30 the cells for 30 seconds at 42 C without the vial(s) from the 42 C bath and place them on ice for 2 add 250 L of pre-warmed Medium to each the vial(s) tightly and shake horizontally at 37 C for 1 hour at 225 rpm in ashaking 20 200 L from each transformation on a pre-warmed selective plate andincubate overnight at 37 C. We recommend that you plate two different volumesto ensure that at least one plate will have well-spaced colonies.

7 For the pUC19control, dilute the transformation mix 1:10 into LB Medium ( remove 100 Lof the transformation mix and add to 900 L of LB Medium) and plate 25-100 the remaining transformation mix at 4 C. Additional cells may be platedout the next day, if the selective plate(s) and incubate at 37 C colonies and analyze by plasmid isolation, PCR, or Shot TOP10 Chemically Competent E. coli Product Information Sheet 3 Transform Competent cells (rapid transformation procedure)This procedure is only recommended for transformations utilizing ampicillin is essential to pre-warm selective plates prior to spreading the transformed the ligation reactions briefly and place on , on ice, one 50 L vial of One Shot cells for each 1 5 L of each ligation reaction into the vial of Competent cells and mix bytapping gently.

8 Do not mix by pipetting up and down. Store remaining ligationreactions at 20 the cells on ice for 5 the cells for 30 seconds at 42 C without the vial(s) from the 42 C bath and place them on ice for 2 spread 50 L of transformed cells on a pre-warmed selective platecontaining 100 g/mL at 37 C colonies and analyze by plasmid isolation/restriction, PCR, or product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at If you have any questions, please contact Life Technologies at information in this guide is subject to change without THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S)

9 WILL NOT BELIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIALDAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF Licensing Information: These products may be covered by one or more Limited Use LabelLicenses. By use of these products, you accept the terms and conditions of all applicable Limited UseLabel entity: Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of ThermoFisher Scientific and its subsidiaries unless otherwise support visit or email October 2015