Editing Protein Expression using Vmax (section)

== Transformation ==

1. Aliquot 5 ul cells into autoclaved&nbsp;microcentrifuge tubes, store at -80 oC.

2. Aliquot 70 ul Vmax™ Chemicompetent Cell Recovery Medium into&nbsp;autoclaved microcentrifuge tubes

3. Take 1 vial of cells&nbsp;out, thaw one vial of Vmax™ Express Chemically Competent Cells on ice for 5–10 minutes. Do not vortex or pipette up and down. If necessary, gently flick the tube to verify that the cells are thawed. Do not transfer cells to another tube; perform heat shock in the tube provided. Note: Use cells within 30 minutes of thawing. 

4. As soon as the chemically competent cells are thawed, add 1–100 ng or 0.5&nbsp;ul of plasmid DNA (in TE buffer or water only) to the competent cells on ice. Briefly mix by gently flicking tubes and immediately place the tubes back on ice. Avoid introducing bubbles and do not vortex or pipette up and down. Note: Add no more than 1 μL of plasmid DNA to the competent cells. Concentrate the plasmid DNA if necessary. 

5. Incubate the DNA and cell mixture on ice for 1 hour. Note: DNA and cells can be incubated for as little as 20 minutes. However, we recommend the full hour incubation because transformation efficiency decreases by ~2-fold for every 15-minute incubation time reduction. 

6. During incubation, warm up 1 vial of 70&nbsp;uL Vmax™ Chemicompetent Cell Recovery Medium.&nbsp;Allow the medium to warm to 30°C or 37°C in a water bath or incubator until needed in Step 9. 

Note: Vmax™ Chemicompetent Cell Recovery Medium is very rich and prone to contamination. Use good aseptic handling practices to avoid contamination. We recommend aseptically transferring an aliquot of media to a sterile tube if the entire bottle will not be used. Store remaining media at 4°C.

7. After incubating cells and DNA on ice, heat shock the Vmax™ Express transformation reactions by transferring them to a '''42°C''' water bath without shaking for '''30 '''seconds. 

8. Immediately transfer the Vmax™ Express transformation reaction back to the ice bucket for 2 minutes. Do not mix. 

9. While keeping the tube on ice, transfer the cells to a pre-warmed &nbsp;tube with Chemicompetent Cell Recovery Medium&nbsp;

10.&nbsp;Place the tubes in an orbital shaker (250 rpm) at 37°C for 2&nbsp;hours to allow cells to recover and express the antibiotic resistance marker. 

Note: Recovery can be performed at 37°C, but 30°C is also acceptable for all antibiotics. 

'''Kanamycin vector-containing cells can be recovered at 37°C or 30°C, but plates must be incubated at 30°C or room temperature'''. It is important to allow the Vmax™ Express cells to recover for 2 hours. Shorter recovery times negatively impact transformation efficiency. 

11. During cell recovery, pre-warm LB-Miller agar plates prepared with antibiotics at the appropriate incubation temperature for your vector/antibiotic selection approach.&nbsp;

12.&nbsp;Transfer all of the transformation reaction to the center of an LB-Miller plate supplemented with the appropriate antibiotic. Use a sterile spreader or sterile beads to distribute cells across the surface of the plate.

Note: If too few colonies are observed, use more&nbsp;&nbsp;DNA&nbsp; for &nbsp;transformation reaction. 

13. Incubate plates at 25°C, 30°C, or 37°C for at least 8 hours.

'''Note:''' For kanamycin plates, incubate at 25°C to 30°C. Do not incubate kanamycin plates at 37°C.&nbsp;

Vmax™ Express has a naturally occurring resistance to kanamycin. Thus, the minimum inhibitory concentration (MIC) for kanamycin selection is higher than that of other laboratory bacteria. Using kanamycin selection with Vmax™ Express, positive clones appear larger and non-transformed naturally resistant colonies appear smaller. DO not use small colonies



'''Important notes'''

• Vmax™ cells on agar plates should be incubated at 25°C to 30°C overnight. If desired, plates can be incubated overnight at 37°C, except for those containing kanamycin, which must be incubated at 25°C to 30°C. 

• Colonies typically appear after 12–16 hours at 25°C to 30°C and after ~8 hours at 37°C. 

• Store agar plates with established Vmax™ Express at room temperature for up to 72 hours. Well-spaced single colonies will remain isolated at room temperature for several days.

• Do not store agar plates containing Vmax™ cells at 2°C to 8°C as this can adversely impact re-propagation and growth. 

• For long-term storage, we recommend preparing glycerol stocks with 25% glycerol and storing at −80°C.

• Plasmid vectors isolated from E. coli tend to have a lower transformation efficiency than those isolated from Vmax™ cells. If needed, plate a larger volume of cells or transform with a larger amount of plasmid. 

• Typical E. coli miniprep protocols are compatible with Vmax™ cells.

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