TITLE: High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the High Efficiency Transformation Protocol for C3019H and C3019I cells. 

[GUIDELINES]
Transformation Protocol Variables

Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.

Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.

Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42°C is optimal.

Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.

Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

DNA Contaminants to Avoid
ContaminantRemoval MethodDetergentsEthanol precipitatePhenolExtract with chloroform and ethanol precipitateEthanol or IsopropanolDry pellet before resuspendingPEG*Column purify or phenol/chloroform extract and ethanol precipitateDNA binding proteins (e.g. Ligase)Column purify or phenol/chloroform extract and ethanol precipitate


[STEPS]
1. Please select whether you have C3019H or C3019I cells.
3. Add 1 µL-5 µL containing 1 pg-100 ng to the cell mixture.
4. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
5. Place the mixture on ice for 30 min. Do not mix. 
 

6. Heat shock at exactly 42 °C for exactly 30 s. Do not mix. 
 

7. Place on ice for 5 min. Do not mix. 

8. Pipette 950 µL into the mixture. 
 

9. Place at 37 °C for 60 min, shaking vigorously (250 rpm) or rotating. 
 

10. Warm selection plates to 37 °C.  
 
11. Mix the cells thoroughly by flicking the tube and inverting.
12. Perform several 10-fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.
 
13. Spread 50 µL-100 µL of each dilution onto a selection plate.
14. Incubate 60 min at 37 °C.
 

2. Thaw a tube of NEB 10-beta Competent E. coli cells on ice for 10 min.
 


2. Thaw a tube of NEB 10-beta Competent E. coli cells on ice until the last ice crystals disappear.
 


3. Mix gently and carefully pipette 50 µL into a transformation tube on ice. 
4. Add 1 µL-5 µL containing 1 pg-100 ng to the cell mixture.
5. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
6. Place the mixture on ice for 30 min. Do not mix. 
 

7. Heat shock at exactly 42 °C for exactly 30 s. Do not mix. 
 

8. Place on ice for 5 min. Do not mix. 

9. Pipette 950 µL into the mixture. 
 

10. Place at 37 °C for 60 min, shaking vigorously (250 rpm) or rotating. 
 

11. Warm selection plates to 37 °C.  
 
12. Mix the cells thoroughly by flicking the tube and inverting.
13. Perform several 10-fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.
 
14. Spread 50 µL-100 µL of each dilution onto a selection plate.
15. Incubate 60 min at 37 °C.