Transforming chemically competant bacteria

9/21/05

 

 

1. Bacteria can be made competant for uptake of DNA through chemcal or electrical transformation. Make sure your cells are chemically competant before following this protocol.

 

2. For difficult cloning and viral vectors, use SURE cells (Stratagene) or Top10 cells (Invitrogen). These are recombination deficient and will be less likely to cause mutations of the plasmid. For routine cloning or transformation of supercoiled plasmid DNA for propogation, use XL1 or DH5alpha cells.

 

3. Cells purchased from a vendor will have an efficiency of 108-109 colonies per ug DNA. Homemade cells will be 106-108.  Purchased cells are expensive so use as little as possible per transformation. 10-20uL should be sufficient. Homemade cells are calibrated for 100uL aliquots. You cannot refreeze any cells you do not use, so make sure you calculate how much you need before thawing.

 

4. Remove competant bacteria from the –80 freezer and place immediately on ice. Thaw on ice. This will take about 20 minutes. In the meantime, remove your plates from the cold and warm up on your bench upside down.

 

5. Place 2-5uL of your ligation or up to 100ng plasmid DNA in a labeled Eppendorf tube and prechill on ice. If you are concerned about the transformation protocol or the competancy of the cells, do a positive control transformation with 100ng of plasmid DNA.

 

6. Add bacteria to the tube and mix very gently.

 

7. Keep on ice 20Õ.

 

8. Heatshock in a 42 degree heatblock for 30 seconds.

 

9. Return to ice,  transfer to a labeled Falcon 2059 tube, and add 250uL LB. (For higher efficiency, use SOC instead of LB). You can skip the recover steps (10-11) if you are just transforming plasmid,.

 

10. Recover with shaking for 1 hour at 37 degrees. Meanwhile, prewarm the agar plates to 37 degrees.

 

11. For ligations plate the entire transformation on one plate. Make sure to use plates containing the correct antibiotic to select for replication of the plasmid you are transforming.

 

12. For plasmids, plate only up to 50uL of the transformation.

 

13. Spread evenly on plate with spreader, then incubate upside down (to prevent condensation from dropping on the plate and spreading the colonies) at 37 degrees overnight.