Vmax™ Express Electrocompetent Cells QRM for Experienced Users
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Vmax™ Express Electrocompetent Cells Quick Reference Manual for Experienced Users Catalog Numbers CL1100-05, CL1100-10, CL1100-20 For complete product information and a detailed protocol visit sgidna.com/vmax-express. Vmax™ Express Electrocompetent Cells Contents Cat. No. CL1100-05 Cat. No. CL1100-10 Cat. No. CL1100-20 Storage Component (5 Reactions) (10 Reactions) (20 Reactions) Temperature* Quantity Vmax™ Express 5 vials 10 vials 20 vials −80°C Electrocompetent Cells Vmax™ Recovery Medium 5 vials 10 vials 20 vials 4°C (after opening) Positive control 1 vial 1 vial 1 vial −20°C pACYC/Chlor plasmid *All components are shipped on dry ice and may be stored at −80°C if desired. Unopened vials of Vmax™ Recovery Medium can be stored at room temperature (20 to 24°C). Vmax™ Express handling practices 6. Transfer cells to a chilled cuvette using a pipette. • Use cells within 6 months of receipt. 7. Transfer the cuvette to the shock pod and perform • Protect cells from temperature fluctuations during electroporation. storage. Note: Time constants typically range from 3.3–3.7 ms. • Thaw cells on ice just prior to use. 8. Transfer cells to a room temperature Falcon® 14-mL • Gently flick tubes to mix or resuspend. Round-Bottom Tube containing recovery media • Pre-warm media before use. immediately after pulse. • After transformation, recover cells for a full 2 hours in 9. To transfer any remaining cells, add 200 μL of Vmax™ the provided Vmax™ Recovery Medium before plating. Recovery Media to the cuvette. Dispense the media to • Store agar plates with Vmax™ Express cells at room the same round-bottom tube from Step 8. temperature. Do not store agar plates at 2°C to 8°C. 10. Place the Falcon® 14-mL Round-Bottom Tubes in a • Agar plates may be left at room temperature for up to tube rack in an orbital shaker (250 rpm) at 30°C or 37°C 72 hours. for 2 hours. • For long-term storage, we recommend preparing 11. During cell recovery, pre-warm LB-Miller agar plates glycerol stocks with 25% glycerol and storing at −80°C. prepared with antibiotics at an appropriate incubation temperature. Electroporation protocol 1. For each transformation reaction, Plating instructions • Thaw one vial of Vmax™ Express 1. Add 50 μL (for vectors isolated from Vmax™ Electrocompetent cells on ice for 5 minutes. cells) or 200 μL (for E. coli-derived vectors) of the • Warm one vial of Vmax™ Recovery Media to room transformation reaction to the center of an LB- temperature (20°C to 24°C). Miller plate. Use a sterile spreader or sterile beads to • Pre-chill a 0.1-cm electroporation cuvette on ice. distribute cells across the surface of the plate. 2. Transfer 500 μL Vmax™ Recovery Media to a Falcon® 2. (Optional) For the control plasmid, dilute the 14-mL Round-Bottom Tube. transformation reaction 1:200 with Vmax™ Recovery 3. As soon as the electrocompetent cells are thawed, add Medium. Plate 50–100 μL of the diluted reaction on an 1–100 ng (2 μL maximum volume) of plasmid DNA (in LB-Miller agar plate supplemented with 5–12.5 μg/mL TE buffer or water only) to the competent cells on ice. chloramphenicol. Briefly mix by flicking tubes. 3. Incubate plates at 25°C, 30°C, or 37°C for at least 4. Incubate the DNA and cell mixture on ice for 8 hours. 3–5 minutes. Note: For kanamycin plates, incubate at 25°C to 30°C. 5. Set the Gene Pulser® Xcell™ System to the following Do not incubate kanamycin plates at 37°C. electroporation parameters: 900 V, 25 uF, 200 Ohms, 0.1-cm cuvette. Note: Use of an electroporator other than the Gene Pulser® Xcell™ System may require optimization of electroporation conditions. Synthetic Genomics, Inc. syntheticgenomics.com Preparing seed cultures Maintaining antibiotic selection 1. Pick the first/largest colonies to appear. Recommended concentrations for maintaining antibiotic 2. Inoculate a culture tube containing 2–15 mL growth selection using Vmax™ Express culture on solid or liquid media supplemented with appropriate antibiotics at culture are listed in the following table. room temperature, 30°C, or 37°C. Solid media Liquid culture Antibiotic marker 3. Culture cells at 30°C or 37°C at 250 rpm according to the Concentration guidelines in the following table. Ampicillin 10–50 μg/mL 50–100 μg/mL Rapid expression Standard Carbenicillin 2–25 μg/mL 5–100 μg/mL protocol protocol Kanamycin 100 μg/mL 200 μg/mL ~3 hours Incubation duration 6–16 hours (for 25 mL of medium) Tetracycline 2.5 μg/mL 2.5 μg/mL Chloramphenicol 5–12.5 μg/mL 12.5–25 μg/mL OD600 post-incubation ≥0.3 2–7 Seed stock use medium-scale large-scale For glycerol stocks? No Yes Recipes For optimal cell growth in liquid cultures, Vmax™ Enriched Glycerol stocks Growth Medium (SGI-DNA Cat. No. CL1500-1000) is recommended for best results. Alternatively, the following 1. Add 250 μL of sterile 100% glycerol to a 1-mL liquid media can be used. For best results use a growth cryopreservation vial or other −80°C compatible sterile media listed here. Other liquid media can result in poor tube. Add 750 μL of bacterial seed culture (prepared as growth and/or expression. described above) and mix for a final concentration of 25% glycerol. Liquid culture Enhanced 2xYT medium 2. Place vials in –80°C freezer for storage. 20 g/L Yeast Extract 3. To recover culture, use a sterile inoculation loop to 32 g/L Tryptone scoop frozen culture and transfer to liquid or solid 17 g/L NaCl growth medium with appropriate antibiotics and supplemented with 0.2% Glucose and 17.6 mM Na2HPO4 incubate at room temperature, 30°C, or 37°C. adjusted to pH 7.4 Note: Always incubate kanamycin-resistant strains Brain heart infusion broth + v2 salts grown on solid agar medium at 30°C or room 37 g/L Brain Heart Infusion Broth Dry Media temperature. (Teknova Cat. No. B9500) supplemented with additional salts: 204.0 mM NaCl Protein expression protocol 4.2 mM KCl 23.14 mM MgCl2 1. Inoculate a seed culture (OD600 of 2–7 for standard protocol, >0.3 for rapid protocol) into growth medium For optimal cell growth on agar plates, we recommend containing appropriate antibiotics. Use 1/100 volume of LB-Miller agar: seed culture relative to the total amount of medium. Solid media 2. Grow culture at 30°C for ~1–2 hours on a rotating shaker LB-Miller agar incubator at 250 rpm until the OD600 reaches >0.5. 10 g/L Tryptone 5 g/L Yeast Extract 3. After reaching OD600 of ≥0.5, induce protein expression by adding IPTG to a final concentration of 1 mM. 10 g/L NaCl 1.5% agar 4. Incubate induced cells for 4–24 hours. 5. Harvest cells by transferring to a centrifugation tube and spinning at 4000–5000 × g for 10 minutes at room temperature or 4°C. Carefully remove supernatant. 6. Store the cell pellet at −80°C or proceed to lysis and protein purification. Complete product information and additional resources are available at sgidna.com/vmax-express. Technical Services [email protected] Trademarks Synthetic Genomics® is a registered trademark and Vmax™ is a trademark of Synthetic Genomics Inc. Gene Pulser® is a registered trademark and Xcell™ is a trademark of Bio-Rad Laboratories, Inc. Falcon® is a registered trademark of Corning Inc. Disclaimer The material in this manual is for informational purposes only and is subject to change without prior notice at any time. Synthetic Genomics and/or its affiliates assume no responsibility for any errors that may appear in this document. Label License Research Vmax™ Express Electrocompetent Cells (Catalog Numbers CL1100-05, CL1100-10, CL1100-20), and components and products thereof, are to be used for Use Only research purposes for the sole benefit of the purchaser only. These cells are sold only for the intended purpose of recombinant protein expression for research purposes. They may not be used for any other purpose, including, but not limited to, use in drugs, diagnostics, therapeutics or in humans. The above mentioned products and components and products thereof, may not be reverse engineered, remade, transferred or sold to third parties, resold, modified for resale, or used to manufacture commercial products or provide commercial services. Except as otherwise agreed in writing by our authorized representative, this product is for INTERNAL RESEARCH USE ONLY AND NOT FOR HUMAN, ANIMAL, THERAPEUTIC OR DIAGNOSTIC USE. For additional information about your rights under this research license, please see our General Terms of Service (located at customer.sgidna.com/TOS.pdf). For information on obtaining additional rights, please contact Synthetic Genomics® at [email protected]. © 2018 Synthetic Genomics Inc. All rights reserved. Part Number 40031 Synthetic Genomics, Inc. REV 1.0 06.08.18.