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From 2010.igem.org

Contents 1 Johan 1.1 Results from yesterdays transformation 1.2 Troubleshooting 1.3 Transformation 1.4 Troubleshooting 1.5 Transformation

Johan

Results from yesterdays transformation

1. Bl21 cells, linear mutagenesis vector, our amp plate: zero colonies
2. Bl21 cells, linear mutagenesis vector, commercial carbenicillin plate: zero colonies
3. Bl21 cells, commercial control vector, our amp plate: zero colonies
4. Bl21 cells, commercial control vector, commercial carbenicillin plate: zero colonies

Troubleshooting

The problem was discussed to be that I used Bl21 cells (used for protein expression) instead of DHα cells (used for cloning), as Bl21 probably don't have the necessary parts for nick ligation that turns the linear vector into a circular vector. However, it could not be satisfying answered why the control plasmid didn't make any colonies.

Transformation

A transformation was set up not using Bl21 cells.

Notes on protocol:

Following constructs was set up:

1. Commercial NEB 5-α cells, 0,5 µl linear mutagenesis vector, SOC medium
2. Commercial NEB 5-α cells, 2 µl linear mutagenesis vector, SOC medium
3. Commercial NEB 5-α cells, 1 µl commercial control vector, LB medium
4. Commercial NEB 5-α cells, 1 µl commercial control vector, SOC medium
5. Our DHα cells, 1 µl commercial control vector, SOC medium
6. Our DHα cells, 1 µl linear mutagenesis vector, SOC medium

Step 10. 50 µl of undiluted cells were plated on carbenicillin plates.

Troubleshooting

The water bath used for heat chock didn't feel warm enough even though it displayed 42 °C, and thermometers didn't go above 38 °C. After a long investigation with prof. Rob it was shown that the "high calibration point" used to give accurate measurements had been decreased. Restoring to factory calibration allowed the water to be heated to 42 °C again. The length of this error is unknown (possible source of error for earlier failed transformations).

Transformation

A new transformation was set up now when proper heat chocks was possible.

Notes on protocol:

1. Commercial NEB 5-α cells, linear mutagenesis vector
2. Commercial NEB 5-α cells, commercial control vector

Step 2. 1 µl was added. Step 10. 50 µl of undiluted cells wewre plated on carbenicillin plates.