Difference between revisions of "Team:Pasteur Paris/Test"

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 Midiprep for plasmid extraction protocol here )
Measure of the DNA concentration in each tube thanks to the 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
260/280	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
260/280 [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.

Transformation:
We received from Eurofins our first sequences:

• 3a_NGF construction Part1 (Seq1)
• 3a_NGF construction Part2 (Seq2)
• T7 RIP construction (Seq8)

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):

• 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.

(See here the liquid culture protocol)

07.20.2018

Results of the liquid culture are good.

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 Midiprep for plasmid extraction protocol here )
Measure of the DNA concentration in each tube thanks to the 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/uL [NA]	260/280
3569.2	1.9
1578.7	1.89
848.1	1.90
2322.1	1.89

For NGF part1, Colony 2:

ug/uL [NA]	260/280
488.1	1.89
493.7	1.90
445.1	1.89
644.6	1.86

For NGF part2, Colony 1:

ug/uL [NA]	260/280
1555.0	1.88
493.7	1.88
666.2	1.87
367	1.90

For NGF part2, Colony 2:

ug/uL [NA]	260/280
463.7	1.89
420.8	1.88
461.4	1.89
420.6	1.89

For RIP, Colony 1:

ug/uL [NA]	260/280
694.4	1.84
1028.9	1.87
1408.7	1.88
466.8	1.88

For RIP, Colony 2:

ug/uL [NA]	260/280
228.4	1.89
325.2	1.89
379.4	1.89
427.2	1.89

Remarks:
[NA] < 0 = no DNA
[NA] > 200 good
260/280 [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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