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Team:Macquarie Australia/Protocols/Making competent Cells
From 2012.igem.org
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| - | 1) Obtain competent E coli cells from -80 C. | + | *'''1) Obtain competent E coli cells from -80 C. |
| - | 2) Hold the tubes of competent cells in your fingers until they are just defrosted, and then put them on ice immediately. 100 µl is sufficient for 2 transformations. | + | *'''2) Hold the tubes of competent cells in your fingers until they are just defrosted, and then put them on ice immediately. 100 µl is sufficient for 2 transformations. |
| - | 3) Add 1-10 µl of ligation mix to each tube. Incubate on ice for 5 min. | + | *'''3) Add 1-10 µl of ligation mix to each tube. Incubate on ice for 5 min. |
| - | 4) Put the tubes in the 42°C bath for 30 sec and put back on ice for 2 min. | + | *'''4) Put the tubes in the 42°C bath for 30 sec and put back on ice for 2 min. |
| - | 5) Add 200 µl of SOC media to each tube, and incubate in the 37oC shaker for 10 min or up to 1hr. I do 10 minutes if it is just a plasmid or 1 hour for ligation mixes. | + | *'''5) Add 200 µl of SOC media to each tube, and incubate in the 37oC shaker for 10 min or up to 1hr. I do 10 minutes if it is just a plasmid or 1 hour for ligation mixes. |
| - | 6) For each tube of cells, spread 5 µl onto one LB (Ampicillin) plate and 50 µl onto a second LB (Ampicillin) plate, using aseptic technique. This should give you a total of four plates. | + | *'''6) For each tube of cells, spread 5 µl onto one LB (Ampicillin) plate and 50 µl onto a second LB (Ampicillin) plate, using aseptic technique. This should give you a total of four plates. |
| - | 7) Place your plates upside-down in the 37°C incubator. | + | *'''7) Place your plates upside-down in the 37°C incubator. |
Revision as of 08:51, 15 August 2012
Biobricks
Methods:
Transformation of E. coli competent cells Experimental procedure:
- 1) Obtain competent E coli cells from -80 C.
- 2) Hold the tubes of competent cells in your fingers until they are just defrosted, and then put them on ice immediately. 100 µl is sufficient for 2 transformations.
- 3) Add 1-10 µl of ligation mix to each tube. Incubate on ice for 5 min.
- 4) Put the tubes in the 42°C bath for 30 sec and put back on ice for 2 min.
- 5) Add 200 µl of SOC media to each tube, and incubate in the 37oC shaker for 10 min or up to 1hr. I do 10 minutes if it is just a plasmid or 1 hour for ligation mixes.
- 6) For each tube of cells, spread 5 µl onto one LB (Ampicillin) plate and 50 µl onto a second LB (Ampicillin) plate, using aseptic technique. This should give you a total of four plates.
- 7) Place your plates upside-down in the 37°C incubator.
