Team:UESTC-China/Protocol

From 2014.igem.org

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           2) Allow cells to grow at 37℃ overnight;<br>
           2) Allow cells to grow at 37℃ overnight;<br>
           3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br>
           3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br>
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           4) Take 2 ml LB media and save for blank, Transfer 5 ml overnight DH5α culture into 500 ml LB media in 3 L flask;<br>
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           4) Take 2 ml LB media and save for blank, transfer 5 ml overnight DH5α culture into 500 ml LB media in 3 L flask;<br>
           5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours);<br>
           5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours);<br>
           6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min;<br>
           6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min;<br>
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           7) Centrifuge cells in at 4oC for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room;<br>
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           7) Centrifuge cells in at 4℃ for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room;<br>
           8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min;<br>
           8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min;<br>
           9) Centrifuge cells at 4O℃ for 10 min at 3,000 g;<br>
           9) Centrifuge cells at 4O℃ for 10 min at 3,000 g;<br>
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           10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol Transfer 140 ml into (1.5 ml) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months;<br>
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           10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol Transfer 140 ml into (1.5 ml) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80℃ can be used for transformation for up to ~6 months;<br>
           11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br>
           11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br>
           12) Incubate the mixture on ice for 30 minutes;<br>
           12) Incubate the mixture on ice for 30 minutes;<br>

Revision as of 12:16, 17 October 2014

UESTC-China