Team:Macquarie Australia/Protocols/Making competent Cells

From 2012.igem.org

(Difference between revisions)
(Methods:)
(Methods:)
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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.