Team:Stockholm/15 July 2010

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Latest revision as of 10:48, 26 October 2010

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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.

Andreas

Protein expression of BBa_J18930-32 from pEX

1. ON cultures from 14/7 were used to inoculate new cultures for IPTG induction:
   • 80 ml LB + 1 % glucose + 100 μg/ml Amp
   • 0.8 ml from ON culture
     • BL21 - pEX.BBa_J18930 (a)
     • BL21 - pEX.BBa_J18931 (a)
     • BL21 - pEX.BBa_J18932 (a)
2. Cultures grown in 37°C, 250 rpm until an OD₆₀₀ of ≈0.5
3. 1 ml samples were extracted from each culture, cells pelleted and resuspended in 50 μl SDS-PAGE sample buffer and stored in -20°C.
4. Remaining culture induced with 0.24 ml 0.1 M IPTG and grown for 2 more hours.
5. New samples (0.5 ml) were extracted, pelleted and cells dissolved in 50 μl SDS-PAGE sample buffer and stored in -20°C.
6. Cultures were checked for fluorescence on the UV table.

No fluorescence was observed for the cells, indicating that something must have gone wrong with the IPTG induction.

BL21 glycerol stocks

Continued from 14/7

1.6 ml from each of the BL21 ON cultures was mixed with 0.4 ml 99 % glycerol and stored in -80°C:

• pEX.BBa_J18930 A
• pEX.BBa_J18930 B
• pEX.BBa_J18931 A
• pEX.BBa_J18931 B
• pEX.BBa_J18932 A
• pEX.BBa_J18932 B

Plasmid prep from Top10 ON cultures with pEX constructs

Continued from 14/7

Plasmids were prepared from ON cultures. Plasmids eluted with 70 μl sterile dH₂O. DNA concentrations were not measured (will be done later). Samples stored in -20°C.

Gel verification of colony PCR samples from 14/7

Johan ran a colony PCR for me of all pSB1A3-, pSB1C3- and pSB1K3-carried constructs (in total 26 samples):

• pSB1A3.BBa_J18930 A/B
• pSB1A3.BBa_J18931 A/B
• pSB1A3.BBa_J18932 A/B
• pSB1A3.SOD A/B

• pSB1K3.BBa_J18930 A/B
• pSB1K3.BBa_J18931 A/B
• pSB1K3.BBa_J18932 A/B
• pSB1K3.SOD A/B

• pSB1C3.BBa_J18930 A/B
• pSB1C3.BBa_J18931 A/B
• pSB1C3.BBa_J18932 A/B
• pSB1C3.SOD A/B
• pSB1C3.yCCS A/B

I also repeated the gel from 14/7 of all pEX constructs (18 samples). 2 gels were prepared and run in parallel.

• 1 % agarose, 80 V, 1 h
• 1 % agarose, 80 V, 45 min

Results

For some reason the samples run by Johan all came out as smears, so the tubes were discarded.