Team:Pasteur Paris/Test
Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
07.16.2018
We transformed pET 43.1a and pSB1C3 in DH5-α competent cells, in order to constitute a stock of empty vectors for our manipulation.
| Plasmid | C (ng/μl) | Volume (μl) | Competent cell | Medium |
| pET 43.1.a | 4.95 | 1 | DH5-α | LB/carbenicilline |
| pSB1C3 | 40 | 1 | DH5-α | LB/chloramphenicol |
See here the transformation of E. coli DH5-alpha protocol We let the transformed bacteria grow overnight (16 hours).
07.17.2018
Results: We went to see our bacterial culture:
- Bacteria transformed with pET 43.1 had not grown.
- Bacteria transformed with pSB1C3 had formed colonies.
Interpretations: Bacteria transformed with pET 43.1 did not grow. We found a non-commercial tube of pET 43.1 in the freezer from last year team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. Transformation did not work as expected, probably because:
- There was no DNA left in the tube.
- The concentration of DNA was too low .
We cultivated the transformed pSB1C3 bacteria in liquid medium 2 x 25ml + Chloramphenicol (25µg/ml) overnight at 37°C, 180 rpm.
Results: Bacteria successfully transformed with pSB1C3. (See here the liquid culture protocol)
07.18.2018
Extraction: We extracted the pSB1C3 plasmid from the bacterial culture. The protocol used was the Qiagen Plasmid Purification Kit (See protocol here ) Measure of the DNA concentration in each tube by NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)
Results: We used the Nanodrop to quantify the purified DNA.
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Volume (µl) | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
| C (ng/µl) | -7.8 | -8.5 | 54.8 | 5.6 | 237.4 | -7.9 | -7.2 | -7.5 | -6.9 | -6.5 |
| Abs(260 nm)/Abs(280 nm) | 1.43 | 1.42 | 1.97 | 3.04 | 1.85 | 1.32 | 1.47 | 1.56 | 1.35 | 1.44 |
Remarks: [NA] < 0 = no DNA [NA] > 200 good Abs(260 nm)/Abs(280 nm) [1;2]
Interpretations:
- Tube 1, 2, 6, 7, 8, 9 and 10 did not contain DNA at all. This problem can be due to the fact that we failed the precipitation with ethanol, meaning that when we removed all the supernatant, we also removed the DNA, or that we removed by mistake the DNA pellet when removing all the supernatant after precipitation (the pellet was so small that we couldn’t see it with the naked eye).
- Tube 3, 4 and 5 had DNA inside. The amount of DNA obtained is enough for us. These tubes have been placed in the freezer for later use.
- 3a_NGF construction Part1 (Seq1)
- 3a_NGF construction Part2 (Seq2)
- T7 RIP construction (Seq8)
- One in an Erlenmeyer in 25 ml LB medium + Carbenicillin (100µg/ml) overnight at 37°C, 180 rpm.
- One in two Falcon tube in 15 ml LB medium + Carbenicillin (100µg/ml) overnight at 37°C, 180 rpm.
Transformation: We received from Eurofins our first gene synthesis DNA products:
In order to constitute a stock of this commercial vectors, we transformed DH5-α competent cells with the plasmid we received, and then we cultivated them on agar plates covered with LB medium and carbenicillin. As the bacteria transformed with pET 43.1a did not grow, we tried again to transform the bacteria with pET 43.1a in a higher quantity than previously.
| Plasmid | C (ng/μl) | Volume (μl) | Competent cell | Medium |
| pEX-A258 Seq1 | 10 | 1 | DH5-α | SOC/carbenicilline |
| pEX-A258 Seq2 | 10 | 1 | DH5-α | SOC/carbenicilline |
| pEX-A258 Seq8 | 10 | 1 | DH5-α | SOC/carbenicilline |
| pET 43.1a | 4.95 | 5 | DH5-α | SOC/carbenicilline |
See here the transformation of E. coli DH5-alpha protocol
07.19.2018
Results: We went to see our overnight bacterial culture plates:
| SEQ#1 3a_NGF_Part1 | SEQ#2 3a_NGF_Part2 | SEQ#8 T7_RIP | Pet43.1a(+) | |
| Selection | Amp | Amp | Amp | Amp |
| Growth | Yes | Yes | Yes | No |
| Liquid Culture | Yes | Yes | Yes | No |
Interpretations: Transformation of Seq#1 – 3a_NGF_Part1, Seq#2 – 3a_NGF_Part2 and Seq #8 – T7_RIP did work as expected. Transformation of Pet43.1a (+) did not work again. As mentioned before, the tube of pET43.1a (+) was a non-commercial tube that we found in the freezer from last year’s team, and we decided to try to amplify it because we did not have any commercial tubes of pET 43.1. We tried to transform our bacteria twice, with no results, so the most probable hypothesis is that the tube did not contain DNA anymore. After this experiment, we decided to buy a tube pET43.1a(+). We cultivated two different colonies from each plate (seq#1, seq#2, seq#8):
(See here the liquid culture protocol)
07.20.2018
Extraction - MidiPrep: We extracted the 3 plasmids Seq#1 – 3a_NGF_Part1, Seq#2 – 3a_NGF_Part2 and Seq #8 – T7_RIP from the bacterial cultures. The protocol used was the Qiagen Plasmid Purification Kit (See protocol here ) Measure of the DNA concentration in each tube by NanoDrop (Blank used : TE.1) (See the NanoDrop protocol here)
Results: We used the Nanodrop to quantify the purified DNA.
For NGF part1, Colony 1:
| ug/μL [NA] | Abs(260 nm)/Abs(280 nm) |
| 3569.2 | 1.9 |
| 1578.7 | 1.89 |
| 848.1 | 1.90 |
| 2322.1 | 1.89 |
For NGF part1, Colony 2:
| ug/μL [NA] | Abs(260 nm)/Abs(280 nm) |
| 488.1 | 1.89 |
| 493.7 | 1.90 |
| 445.1 | 1.89 |
| 644.6 | 1.86 |
For NGF part2, Colony 1:
| ug/μL [NA] | Abs(260 nm)/Abs(280 nm) |
| 1555.0 | 1.88 |
| 493.7 | 1.88 |
| 666.2 | 1.87 |
| 367 | 1.90 |
For NGF part2, Colony 2:
| ug/μL [NA] | Abs(260 nm)/Abs(280 nm) |
| 463.7 | 1.89 |
| 420.8 | 1.88 |
| 461.4 | 1.89 |
| 420.6 | 1.89 |
For RIP, Colony 1:
| ug/μL [NA] | Abs(260 nm)/Abs(280 nm) |
| 694.4 | 1.84 |
| 1028.9 | 1.87 |
| 1408.7 | 1.88 |
| 466.8 | 1.88 |
For RIP, Colony 1:
| ug/μL [NA] | Abs(260 nm)/Abs(280 nm) |
| 228.4 | 1.89 |
| 325.2 | 1.89 |
| 379.4 | 1.89 |
| 427.2 | 1.89 |
Remarks: [NA] < 0 = no DNA [NA] > 200 good Abs(260 nm)/Abs(280 nm) [1;2]
Interpretations: We have DNA in our tubes. The DNA is too concentrated so we will have to dilute them before next use. We froze the tubes at -20°C until next use.
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
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Microfluidics/Membrane
Product Design
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
Something was done one that day
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
Something was done one that day
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
Something was done one that day
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
Something was done one that day
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
Something was done one that day
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
Something was done one that day
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Bacteriology
Cell culture
Microfluidics/Membrane
Product Design
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