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1. Thaw competent cells on ice (40 µl). You will need 4 tubes, one for each of your experimental samples, one positive control and one negative control (see below).
2. Add 10 µl of your ligated, desalted DNA to the cells; allow them to mix for 1-2 minutes.
3. The negative control consists of cells plus a volume of the water that is equal to the volume of DNA solution you add to your cells (in this case 10 µl). Use the same water that you used to re-suspend your DNA.
4. Whenever you are using freshly prepared cells, you should also run a positive control. For a positive control you transform the cells with a known plasmid at a known concentration. You will use pBluescript KS vector that I have prepared. You will use 10 µl of a 1 picomolar solution. This is a positive control because we know that it is circular DNA in a purified form at a known concentration. If you do not get transformants from this control, you can assume that there is something wrong with your cells, your method or the apparatus. The positive control will also allow you to estimate the transformation efficiency of your cells using the electroporation procedure. Transformation efficiency is defined as the number of colonies formed per µg of DNA. You will use only 10 µl of a 1 picomolar solution. How will you calcuate the transformation efficiency?
5. Set the electroporator apparatus at 1.5 kV.
6. Transfer the cells plus DNA to an electroporation cuvette and tap the suspension to the bottom of the cuvette. Place the cuvette in the holder (chilled), push the holder into the electroporator chamber and press PULSE twice. Wait for the "beep" then remove your cells and immediately add 750 µl of SOC media. Transfer cell plus SOC to a sterile test tube. Wash the cuvette with another 750 µl of SOC and add this to the test tube. Incubate your cells at 37^oC, gently shaking for 1-2 hours.
7. After incubation, transfer the cells plus SOC to a microcentrifuge tube and pop spin, draw off all but ~ 100 µl of the SOC, re-suspend the cells and plate on an LB plus ampicillan/tetracycline plate. Place in the incubator, upside down, overnight. Check cultures after 16 hours. If you have colonies on the plate, place it in the refrigerator until you are ready to start your liquid cultures

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¹ A protocol for heat shock transformation is given below. Cells must be made competent for heat shock transformation before use. A protocol for preparing competent cells can be found in Sambrook et al. (2001).

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HEAT SHOCK TRANSFORMATION

1. Add 2-10 µl of ligated DNA to a vial of thawed competent cells¹ and mix gently. Do not mix by pipetting up and down.
2. Incubate on ice for 30 minutes.
3. Heat shock the cells for 30 seconds at 42^oC without shaking.
4. Immediately transfer the cells to ice.
5. Add 250 µl of room temperature SOC medium.
6. Cap the tube tightly and shake horizontally at 37^oC for 30 minutes.
7. Spread the cells on 2 LB plates containing an appropriate antibiotic.
8. Incubate the plates overnight at 37^oC. Remove the plates from the incubator and store at 4^oC until ready for use.