Sack: Bacteria Transformation (One Shot Top Ten Invitrogen)

Sack and Yarov-Yarovoy Labs

Department of Physiology and Membrane Biology
University of California, Davis

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One Shot® TOP10 Chemically Competent E. coli Cat. Nos. C4040-10 (10 reactions); C4040-03 (20 reactions); Store at -80°C Caution C4040-06 (40 reactions) This product contains irritants and may be harmful if swallowed. Review the Material Safety Data Sheet before handling. Description TOP10 E. coli are provided at a transformation efficiency of 1 x 109 cfu/μg supercoiled DNA and are ideal for high-efficiency cloning and plasmid propagation. They allow stable replication of high-copy number plasmids. The genotype of TOP10 Cells is similar to the DH10BTM strain. Components Supplied TOP10 Chemically Competent E. coli Cells 21 x 50 μl pUC19 Control 50 μl DNA (10 pg/μl) S.O.C. Medium 6 ml Genotype F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacΧ74 recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG λ- General Guidelines Follow these guidelines when using One Shot® TOP10 Chemically Competent E. coli. Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Thaw One Shot® competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by swirling or tapping the tube gently. Do not mix cells by pipetting. One Shot® TOP10 cells do not require IPTG to induce expression from the lac promoter. If blue/white screening is required to select for transformants, make sure that selective plates contain 50 μg/ml X-gal. Transforming Competent Cells Perform the following before starting the transformation procedure: Equilibrate a water bath to 42°C. Warm the vial of S.O.C. Medium (supplied with the kit) and LB Medium (if needed) to room temperature. Spread X-gal onto LB agar plates containing antibiotic, if desired. Warm the selective plates in a 37°C incubator for 30 minutes (use 1 or 2 plates for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100 μg/ml ampicillin. Transformation Procedure Use this procedure to transform One Shot® TOP10 chemically competent E. coli. We recommend including the pUC19 control plasmid DNA supplied with the kit (10 pg/μl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8) in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation. Thaw, on ice, one vial of One Shot® TOP10 chemically competent cells for each transformation. Add1to5μloftheDNA(10pgto100ng)intoavialofOneShot® cellsand mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1 μl) of DNA into a separate vial of One Shot® cells and mix gently. Incubate the vial(s) on ice for 30 minutes. Heat-shock the cells for 30 seconds at 42°C without shaking. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes. Aseptically add 250 μl of pre-warmed S.O.C. Medium to each vial. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator. Spread 20-200 μl from each transformation on a pre-warmed selective plate and incubate overnight at 37°C. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium (e.g. remove 100 μl of the transformation mix and add to 900 μl of LB Medium) and plate 25-100 μl. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired. Invert the selective plate(s) and incubate at 37°C overnight. Select colonies and analyze by plasmid isolation, PCR, or sequencing. Note For a rapid transformation procedure, see the next page. Calculating Transformation Efficiency Use the following formula to calculate the transformation efficiency as transformants (in cfu) per μg of plasmid DNA. Remember that the total volume of the transformation mixture is 300 μl. Transformation efficiency (# transformants/μg DNA) = (# of colonies/10pg pUC19 DNA)x(10^6pg/μg)x(300 μl total voulme/X μl plated)x(dilution factor) For example, if transformation of 10 pg of pUC19 DNA yields 100 colonies when 30 μl of a 1:10 dilution is plated, then the transformation efficiency is: (100 colonies/10 pg DNA)×(10^6pg/μg)x(300 μl total volume/30 μl plated)x(10)=1x10^9 Rapid Transformation Procedure This procedure is only recommended for transformations utilizing ampicillin selection. It is essential to pre-warm selective plates prior to spreading the transformed cells. 1. Centrifugetheligationreactionsbrieflyandplaceonice. 2. Thaw, on ice, one 50 μl vial of One Shot® cells for each transformation. 3. Pipet1-5μl of each ligation reaction into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. Store remaining ligation reactions at -20°C. 4. Incubate the cells on ice for 5 minutes. 5. Heat-shock the cells for 30 seconds at 42° without shaking. 6. Removethevial(s)fromthe42°Cbathandplacethemonicefor2minutes. 7. Immediately spread 50μl of transformed cells on a pre-warmed selective plate containing 100 μg/ml ampicillin. 8. Incubateat37°Covernight. 9. Select colonies and analyze by plasmid isolation/restriction, PCR, or sequencing. Quality Control Each lot of TOP10 competent cells is tested for transformation efficiency using the control plasmid included in the kit and the protocol on page 2. Transformed cultures are plated on LB plates containing 100 μg/ml ampicillin and incubated overnight. Transformation efficiency should be ~1 x 109 cfu/μg plasmid DNA. In addition, untransformed cells are tested for the appropriate antibiotic sensitivity and the absence of phage contamination. ©1997-2006 Invitrogen Corporation. All rights reserved. For research use only. Not intended for any animal or human therapeutic or diagnostic use.