Team:WHU-China/Safety

1.1            Storage

1.        Dried DNA: room temperature

2.        Resuspended DNA: -20℃ freezer

3.        The linearized plasmid backbones (25ng/ul at 50ul) should be stored at 4C or lower

1.2            Usage

1.        With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.

2.       

3.        Add 10uL of diH2O (deionized water), the resuspension will become red due to the cresol red dye used during manufacturing.

4.        Pipette 1 or 2uL of the resuspended DNA transform into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.

5.        Pick a single bacterial colony and transfer it into LB medium. Incubate the culture for 18 hours with vigorous agitation.

6.        Store the single colony in physiological saline and on inclined plane, and make recognizable marks on the tube and on the notebook.

Ⅱ. Preparation of competent cells (Using Calcium Chloride)

2.1  Day 0:

    Pick a single bacterial colony from a plate that has been incubated for 16-20 hours at 37℃. Transfer the colony into LB medium. Incubate the culture overnight (about 16 hours) at 37℃ with vigorous shaking.

2.2  Day 1:

1.        Transfer 1 ml of the culture into 100 ml of LB medium. Incubate the culture for 2.5-3 hours at 37℃ with vigorous agitation (250-300rpm).

2.        Transfer 1.5 ml of the culture into sterile ice-cold 1.5ml polypropylene tubes. Cool the cultures by storing the tubes on ice for 10 minutes.

3.        Recover the cells by centrifugation at 2500 rpm for 5minutes at 4℃.

4.        Decant the medium from the cell pellets. Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M CaCl2 solution. Store the tubes on ice for 20minutes.

5.        Recover the cells by centrifugation at 2500 rpm for 5 minutes at 4℃.

6.        Decant the medium from the cell pellets. Resuspend each pellet by swirling or gentle vortexing in 100ml of ice-cold 0.1M CaCl2 solution.

7.        The suspension of competent cells should be immediately used in transformation.

 

Attention:

[1]     Mix gently

[2]     The newly prepared competent cells should be transformed immediately. They cannot be stored for long (except in -80℃)

[3]     Keep sterile environment during operation (sterilize the clean bench by ultra-violet, sterilize hands and implements by alcohol).

[4]     Use heat-shock method to transform.

 

Sed non urna. Donec et ante. Phasellus eu ligula. Vestibulum sit amet purus. Vivamus hendrerit, dolor at aliquet laoreet, mauris turpis porttitor velit, faucibus interdum tellus libero ac justo. Vivamus non quam. In suscipit faucibus urna.

Nam enim risus, molestie et, porta ac, aliquam ac, risus. Quisque lobortis. Phasellus pellentesque purus in massa. Aenean in pede. Phasellus ac libero ac tellus pellentesque semper. Sed ac felis. Sed commodo, magna quis lacinia ornare, quam ante aliquam nisi, eu iaculis leo purus venenatis dui.

Cras dictum. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia mauris vel est.

Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

Cras dictum. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia mauris vel est.

Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

Cras dictum. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia mauris vel est.

Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

Cras dictum. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Aenean lacinia mauris vel est.

Suspendisse eu nisl. Nullam ut libero. Integer dignissim consequat lectus. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

Count Down