Transformation Protocol: Low Copy:
Post Ligation / Low to Intermediate Plasmid

Purpose - to transform a plasmid of intrest inot a bacterial
cell line, in this case, DH5α Competent cells.

Protocol: Modified from Stratagene manual.

1. Preheat LB/Amp plates in the 37oC incubator.
2. Get a bucket of ice.
3. Remove the DH5α cells stored @ -80oC and thaw on ice.
4. Aliquot 100 mL of competent cells into a chilled 1.5mL microcentrifuge tube.
5. Optional Step: Add 0.4mL of 1:10 diluted BME [also stored in the -80oC freezer]

1. BME makes the cells uptake the plasmid DNA better

6. Add 10.0 mL or 1/10th cell vol. of plasmid DNA.
7. Incubate on ice for 30 minutes.
8. After 30 minute incubation, heat-pulse cells in a 42oC water bath for 45 seconds.
9. Incubate cells on ice for 2 minutes.
10. Then add 1mL or 10 x cell vol. of preheated LB
11. Incubate this reaction at 37oC for 1 hour with shaking (225-250 rpm).
12. After the 1 hour incubation and shaking, centrifuge at 3500 rpm for 2 min. 
13. Remove 960 mL or all but 150 mL of the supernatant.
14. Plate and streak the 150mL of transformed cells onto the pre-warmed agar plates containing antibiotic.
15. Incubate the culture at 37oC overnigh
    
    Note: If a high copy plasmid is used, substitute steps 4,6, and 10 with:
    
    4.    Aliquot  20-25 uL of competent cells into a chilled 1.5mL microcentrifuge tube.
    6.    Add 1.0 uL of plasmid DNA
    10.  Then add 200-250Ul or 10x cell vol. of preheated LB
    
    In this case of using a high copy plasmid, steps 12, 13 and 14 are not needed for the protocol

*Allow cells to grow for 12-16 hours.  Re-streak a single, isolated transformed colony onto a fresh agar plate with antibiotic – streak for isolation.  To store old plates, simply wrap them in parafilm and keep in the large (4oC) cold room.