Preparation of competent cells

Keep the cells and all materials cold throughout procedure!! 

1.                  Innoculate 3 mls LB + antibiotic with a single (fresh O/N) colony

2.                  Innoculate 1 L LB + antibiotic with this 3 ml culture

3.                  Grow cells (37 C/shaking) to 0.5-0.8 OD(600) (cell strain-dependent)

4.                  Chill cells on ice 15-30 min. 

5.                  Spin down cells 15 min. (4K rpm) @ 4 C, remove as much SN as possible

6.                  Suspend cells in 1 L ice cold H2O

7.                  Spin down cells as in step 5. 

8.                  Suspend cells in 0.5 L ice cold H2O

9.                  Spin down cells as in step 5. 

10.             Suspend cells in 20 mls ice cold sterile 10% glycerol

11.             Spin down cells as in step 5. 

12.             Suspend cells in 2.5 mls ice cold sterile 10% glycerol

13.             Quick freeze ~125 ul aliquots on dry ice and store -80 C

Desalting ligation mixes

This method requires minimal manipulation of the DNA, which is a significant advantage for ligation involving large DNA fragments.

1. Fill an Eppendorf tube with 1.2 ml of molten 1% low melting agarose in 100mM glucose.

2. Before gelling, vertically immerse a 200µl plastic micropipette tip into the surface of the agarose.

3. After the agarose solidifies remove the tip to leave a conical shaped well of approximately 30-l00µl volume, depending on how far the tip has been immersed into the agarose.

4. Load the completed ligation reaction into the "well" using a micropipette and incubated for 90 minutes on ice to let the salt diffuse into the agarose.
Carefully remove the liquid mixture from the well, and a portion or all of it is directly electroporated into bacteria.