PCR Transformation

 

1. Grow cells to an OD600 0.7-2.5 (25ml culture)

2. Spin cells 4500 rpm for 2 min

3. Resuspend in 50 ml 0.1M LiOAc

4. Repeat step 2 and 3

5. Resuspend in 0.1M LiOAc (culture volume x OD/100)

6. Add 1/9 volume of 10 mg/ml ssDNA that has been boiled for 10 min and cooled on ice

7. Add 15 ml of PCR product to 30 ml of cells and incubate for 15 min at 30oC

8. Add 150 ml Transformation Goop and incubate for 30 min at 30 oC

9. Heat　shock at 42 oC for 15 min

10. Spin cells at 2000 rpm for 2 min

11. Aspirate PEG and resuspend cells in 200 ml YEPD

12. Incubate in the 30 oC shaker for 4 hours

13. Plate cells on selective medium

 

Transformation Goop:   

1 ml sterile ddH2O

1 ml 0.1M LiOAc

       8 ml filtered PEG (Dissolve 15g of PEG3550 in 16.5 ml ddH2O, filter sterilize)