(using chemically competent cells)
1. Remove competent cells from –80C freezer and thaw in room temperature water. After thawing, place on ice. Thaw SOC medium aliquot. Check to see if the 42C water bath is properly set and filled with sufficient water. If it is low add water after your transformation.
2. Once the cells are thawed, remove 200ml aliquots to separate tubes for transformation. Add DNA. If you are transforming from a ligation I often add the entire ligation to the cells –especially if the ligation was difficult as is the case when using a gel extracted vector. If the ligation was from a non-gel extracted vector I often add only a small portion -like 5ml to the cells.
3. Mix gently by inverting the tubes.
4. Heat Shock the cells:
Take your ice bucket and equipment over to the 42C water bath. Place the tubes in a floating rack and get a timer set it for 2 minutes. Have your SOC thawed with pipet and tips ready sitting next to the water bath. Drop the rack of tubes in the water and start the timer. Do not leave the area and do other things, wait for the timer. Once it goes off immediately remove the tubes from the water bath and place on ice. Set the timer again for 2 minutes and leave on ice. Once the timer goes off, add 500 ml of SOC medium.
5. Place tube in shaker incubator at 37C. Tape the tube on its side to the platform base. Leave in incubator for approximately one hour. If your plasmid is Ampicillin selected you can reduce the time to one half hour.
6. After the incubation is over, spread your cells using a flamed hockey stick on a solid LB plate containing the appropriate antibiotic selection. You should do two plates one with 100ml of your transformation, the other with 10ml of the transformation. This is to ensure that you will have colonies separated enough to be able to be picked. If your ligation efficiency was likely to be very low –as is the case for gel extracted vectors- you can simply put the whole transformation on one plate, place it in the hood for a few minutes to dry.
7. If you plated only part of your transformation reaction save the cells by placing them at 4C. You can re-plate the cells later if you don’t get any colonies the first time or you have too many colonies.
8. Place your plate upside down in the 37C incubator overnight (16hr). Be careful not to leave plates with ampicillin too long or satellite colonies will form.
9. Proceed to minipreps protocol
2% Tryptone
0.5% Yeast Extract
10mM NaCl
2.5mM KCl
10mM MgCl2
10mM MgSO4
20mM Glucose