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Team:Paris Liliane Bettencourt/Notebook/2011/09/02/
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Electroporation of Danyel's S81 in pHM3 seems to be a success. However, the positive controls grew even on 75 μg.ml<sup>-1</sup> without the pHM3 resistance to TetR. This brings about a BIG problem: our wild strain is resistant to high levels of tetracycline either because the tet(l) gene mutated (name of gene might be incorrect but natural tetR resistance gene exists) or the strain we were given was already very resistant. The other possibility is that it was given with a plasmid :: TetR, the last possibility is that our tetracycline is non-functional (photodegradation, others ?). In any case, I doubled and quadrupled concentrations and replated as little biofilm as possible of each of the transformants. | Electroporation of Danyel's S81 in pHM3 seems to be a success. However, the positive controls grew even on 75 μg.ml<sup>-1</sup> without the pHM3 resistance to TetR. This brings about a BIG problem: our wild strain is resistant to high levels of tetracycline either because the tet(l) gene mutated (name of gene might be incorrect but natural tetR resistance gene exists) or the strain we were given was already very resistant. The other possibility is that it was given with a plasmid :: TetR, the last possibility is that our tetracycline is non-functional (photodegradation, others ?). In any case, I doubled and quadrupled concentrations and replated as little biofilm as possible of each of the transformants. | ||
| - | + | <br> | |
In parallel, I am going to test a simple construct to test efficiency | In parallel, I am going to test a simple construct to test efficiency | ||
| - | pVEG-SpoVG-RFP-terminator | + | pVEG-SpoVG-RFP-terminator <br> |
| - | bacterial strains were inoculated today. | + | bacterial strains were inoculated today.<br> |
| + | <big>The september 2nd deadlines have been met! And Grenoble is cooperating with us! </big> | ||
| + | |||
| + | ==Danyel== | ||
| + | ===PCR Colony Results=== | ||
| + | S81 in pHM3:[[File:S81 in pHM3 PCR Colony.jpg|450px|thumb|center|The size of the insert in pHM3 is around 290 bp]] | ||
| + | A glycerol of the 4th clone was made and a culture prepared for a miniprep. | ||
| + | |||
| + | S80 in S24:[[File:S24 PCR Colony.jpg|450px|thumb|center|The size of the insert in S24 is suppose to be around 2.7 kb]] | ||
| + | The results are clear: the ligation was a complete failure. Taking into consideration the innumerable amount of colonies on the negative control plate, the S24 has not been digested properly. Edward had very clean negative controls with S24 as a vector. The only differences were that: 1. He used the newly arrived PstI enzyme 2. He gel extracted the plasmid even if it was digested with SpeI and PstI (so far I have done PCR purification). | ||
| + | |||
| + | ===Digestion=== | ||
| + | S24 was redigested on S & P sites, then gel extracted. | ||
| + | |||
| + | |||
| + | S82 (pHyperspank): one tube was digested on S & P , the other was digested on E & S. Both digestion products were loaded on gel (1% and 2.5% respectively). The gels have been cut and are ready for extraction. | ||
| + | |||
| + | ===Ligation=== | ||
| + | Ligations to do tomorrow: | ||
| + | S24 + S38 | ||
| + | S24 + S80 | ||
| + | S82 + S38 | ||
| + | S82 + S58 | ||
| + | (S14 + S38 | ||
| + | S14 + S58) | ||
| + | A S14 culture was prepared from the glycerol for a miniprep. | ||
| + | |||
| + | ==Mathias & Laura== | ||
| + | |||
| + | ===Miniprep=== | ||
| + | |||
| + | -LacI controled T7 and reporter for Bacillus | ||
| + | |||
| + | -T7 Autoamplifier system + Reporter | ||
| + | |||
| + | -pSB1AK3 K143001 | ||
| + | |||
| + | ===Digestion and gel extraction=== | ||
| + | |||
| + | -LacI controled T7 and reporter for Bacillus by EcoR1 and Pst1 | ||
| + | |||
| + | -T7 Autoamplifier system + Reporter by EcoR1 and Pst1 | ||
| + | |||
| + | -pSB1AK3 K143001 by EcoR1 and Pst1 | ||
| + | |||
| + | ===Ligation and transformation=== | ||
| + | |||
| + | -LacI controled T7 and reporter for Bacillus & pSB1AK3 | ||
| + | |||
| + | -T7 Autoamplifier system + Reporter & pSB1AK3 | ||
Latest revision as of 16:31, 3 September 2011
Contents |
Adrien
Electroporation of Danyel's S81 in pHM3 seems to be a success. However, the positive controls grew even on 75 μg.ml-1 without the pHM3 resistance to TetR. This brings about a BIG problem: our wild strain is resistant to high levels of tetracycline either because the tet(l) gene mutated (name of gene might be incorrect but natural tetR resistance gene exists) or the strain we were given was already very resistant. The other possibility is that it was given with a plasmid :: TetR, the last possibility is that our tetracycline is non-functional (photodegradation, others ?). In any case, I doubled and quadrupled concentrations and replated as little biofilm as possible of each of the transformants.
In parallel, I am going to test a simple construct to test efficiency
pVEG-SpoVG-RFP-terminator
bacterial strains were inoculated today.
The september 2nd deadlines have been met! And Grenoble is cooperating with us!
Danyel
PCR Colony Results
S81 in pHM3:
A glycerol of the 4th clone was made and a culture prepared for a miniprep.
S80 in S24:
The results are clear: the ligation was a complete failure. Taking into consideration the innumerable amount of colonies on the negative control plate, the S24 has not been digested properly. Edward had very clean negative controls with S24 as a vector. The only differences were that: 1. He used the newly arrived PstI enzyme 2. He gel extracted the plasmid even if it was digested with SpeI and PstI (so far I have done PCR purification).
Digestion
S24 was redigested on S & P sites, then gel extracted.
S82 (pHyperspank): one tube was digested on S & P , the other was digested on E & S. Both digestion products were loaded on gel (1% and 2.5% respectively). The gels have been cut and are ready for extraction.
Ligation
Ligations to do tomorrow: S24 + S38 S24 + S80 S82 + S38 S82 + S58 (S14 + S38 S14 + S58) A S14 culture was prepared from the glycerol for a miniprep.
Mathias & Laura
Miniprep
-LacI controled T7 and reporter for Bacillus
-T7 Autoamplifier system + Reporter
-pSB1AK3 K143001
Digestion and gel extraction
-LacI controled T7 and reporter for Bacillus by EcoR1 and Pst1
-T7 Autoamplifier system + Reporter by EcoR1 and Pst1
-pSB1AK3 K143001 by EcoR1 and Pst1
Ligation and transformation
-LacI controled T7 and reporter for Bacillus & pSB1AK3
-T7 Autoamplifier system + Reporter & pSB1AK3
