Bacterial Transformation by Heat Shock
You will need:
Bucket of ice
100ng-500ng (~2 uL) of plasmid DNA to be transformed in a sterile eppendorf tube
Competent bacteria cells (DO NOT THAW until ready to begin)
Heat block set to 42C
LB Broth
Bunsen Burner
Micropipettors and pipette tips
37C water bath
2 LB agar plates with appropriate antibiotic
37C plate incubator
1. Place tube with plasmid DNA and a tube of competent cells on ice.
2. Allow competent cells to thaw on ice. Do not use your hands to speed it up.
3. As soon as they are thawed, add 50-100uL of the cells to the tube containing the DNA.
4. Incubate on ice for 15'.
5. Heat-shock the cells by placing in the 42C heat block for 90 seconds.
6. Return the tube to ice.
7. Incubate on ice for 2'.
8. Add 300uL of sterile LB broth to the tube using sterile technique.
9. Incubate in a 37C water bath for 45'-1 hour.
10. Plate an aliquot of the cells on LB/antibiotic plates.
11. Invert plates and grow at 37C overnight (Not more than 18-20 hours).
Note on plating: If you are transforming a plasmid of known concentration, you will only need to plate 20-30uL. If concentration is unknown, plate 20uL and 120uL. If you are transforming a ligation reaction, you will want to plate more cells, generally around 100-120uL, on multiple plates.
Bucket of ice
100ng-500ng (~2 uL) of plasmid DNA to be transformed in a sterile eppendorf tube
Competent bacteria cells (DO NOT THAW until ready to begin)
Heat block set to 42C
LB Broth
Bunsen Burner
Micropipettors and pipette tips
37C water bath
2 LB agar plates with appropriate antibiotic
37C plate incubator
1. Place tube with plasmid DNA and a tube of competent cells on ice.
2. Allow competent cells to thaw on ice. Do not use your hands to speed it up.
3. As soon as they are thawed, add 50-100uL of the cells to the tube containing the DNA.
4. Incubate on ice for 15'.
5. Heat-shock the cells by placing in the 42C heat block for 90 seconds.
6. Return the tube to ice.
7. Incubate on ice for 2'.
8. Add 300uL of sterile LB broth to the tube using sterile technique.
9. Incubate in a 37C water bath for 45'-1 hour.
10. Plate an aliquot of the cells on LB/antibiotic plates.
11. Invert plates and grow at 37C overnight (Not more than 18-20 hours).
Note on plating: If you are transforming a plasmid of known concentration, you will only need to plate 20-30uL. If concentration is unknown, plate 20uL and 120uL. If you are transforming a ligation reaction, you will want to plate more cells, generally around 100-120uL, on multiple plates.
Comments
Post a Comment