Team:Pasteur Paris/Microbiology week2

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Aim: Check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.
Seed pET43.1 DH5α in LB + carbenicillin media to make stab culture. Indeed, the validity of transformants needs to be verified by checking if an insert is present in the transformed plasmid. Secondly, verified plasmid-DH5&alpha combinations need to be stored beyond the lifetime of plate colonies.

Protocol: follow in this link

What we did in the lab:
Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 µg/ml (LB + CB50) or with chloramphenicol 34 µg/ml (LB + CM34) were counted for the presence of individual distinct colonies.

LB + CB50

	Box 1	Box 2	Box 3
pET43.1a(+) DH5α	0	2	23
pET43.1a(+) DH5α (different batch)	Uncountable (Overgrown)	2
pET43.1a(+) DH5α (different batch)	Uncountable (Overgrown)	15

Table 3
LB + CM34

	Box 1	Box 2
pSB1C3 DH5α	1	78
pSB1C3 DH5α (different batch)	38	Uncountable (Overgrown)

Table 4

What we did in the lab:
Materials:
• 1X TAE buffer
• Agarose
• Microwave oven /
• Distilled water
• Ethidium Bromide
• Gel caster, and power supply
• Precision balance

Method:
1. Buffer solution: we have 1.0X stock TAE (Tris Acetate EDTA) solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer
2. Agarose gel: We prepared 0.7% w/v agarose in 1X TAE and allow it to set.

For agarose gel electrophoresis experiment:

Samples:
• pSB1C3 plasmid (obtained with Midiprep on June 8, 2016)
• pSB1C3 plasmid (digested by SpeI/XbaI)
• pET43.1a plasmid (obtained with Midiprep on June 8, 2016)< /br> • pET43.1a plasmid digested by BamH I/Hind III
• gel 0.7% agarose
• TAE 0.5X buffer
• Electrophoresis power supply
• DNA ladder (Thermofisher Gene ruler 1kb)
)
Method:
1. After filling the electrophoresis chamber with TAE 0.5X buffer, samples were loaded in 1X gel loading buffer in the wells, according to the following lane pattern.

	Lanes	L1	L2	L3	L4-7	L9-8	L10-13	L14	L15
Sample	Molecular weight	pET43.1a(+) uncut		pET43.1a(+) B-H		pBS1C3 S-X		pBS1C3 uncut
	DNA (µl)	5	2		2		4		4
	H20	0	8		8		6		6
	6X Loading buffer	0	2		2		2		2

Table 5

Results
The digestion has worked since the plasmids digested have moved faster.

NB: the gel suffered from overheating.

Figure 1:

Aim: to make a stab culture we need to capture the bacteria growing at the exponential phase
In order to do that we need to take optical density at 600_nm (OD600_nm).

Protocol: follow in this link

What we did in the lab:
Materials:
• Falcon of 50 ml
• LB autoclaved 500 ml
• LB (Luria Broth) autoclaved (500 ml)
&bull Precultures performed on the June 14, 2016
• Antibiotic: carbenicillin 100 mg/ml
• Microbiology equipment

Method:
Before starting the culture we need to have molten LB-agar so we need to place the solid LB agar on a hot plate with stirring.
1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600_nm every hour for the first three hours, then every 20 min onwards. We recorded OD600_nm for this specific experiment after 3 hours.

	Time	Absorbance A’	Absorbance A’’
12h37		0.013	0.015
13h07	0.023	0.027
13h37	0.063	0.059
14h07	0.110	0.105
14h27	0.172	0.170
14h47	0.265	0.256
15h07	0.384	0.377
15h27	0.491	0.482
15h47	0.567	0.557
16h06	0.697	0.608
16h29	0.816	0.755
17h03	0.954	0.909
17h20	0.954	0.909
17h40	1.061	1.029
18h00	1.092	1.082
18h00	1.122	1.132

Table 6

Growth curve :
Figure 2: Growth curve for DH5α-pET43.1a(+) by measurement of OD600_nm. O-time point corresponds to 12h37

Aim: we received our gene block synthesized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer.

Method:
Add TE (Tris 10 mM pH 8.0 EDTA 1 mM) buffer in each tubes and refer to the next table for volumes.

Name	Weight (mg)	Cfinal (ng/µl)	V(TE) (µl)
A1	500	10	50
A2	500	10	50
C1	1000	10	100
C2	1000	10	100
D1	500	10	50
D2	500	10	50
E1	500	10	50
E2	500	10	50
B2	1000	10	100

Table 7

Aim: We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. Spe I/ Xba I or BamH I/Hind III respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN. Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2.

Protocol: follow in this link

What we did in the lab:
Material:
• We use the NEB extraction kit. Monarch DNA Gel Extraction Kit (/#T1020G)

Method:
1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube.
2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8:

		Empty Eppendorf (g)	Eppendorf + gelf (g)	Final weight (mg)
pET43.1a(+)	m1	0.9	1.3	329.6
pET43.1a(+)	m2	0.9	1.3	265.3
pSB1C3	m1	0.9	1.2	251.1
pSB1C3	m2	1.0	1.2	185.6

Table 8

3. Incubate the samples between 37-55°C, vortexing periodically until the gel slice is completely dissolved (/ ~ 10 min)
4. Load sample onto the column. Close the cap, spin for 1 min then discard flow through
5. Re insert column into collection tube. Add 200 µµl DNA wash buffer and spin for one minute. Discard flow-through
6. Repeat step 5
7. Transfer column to a clean 1.5 ml microfuge tube
8. Add 6 µl of DNA Elution buffer to the center of the matric. Wait for 1 min and spin for 1 min to elute DNA

Aim: after digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2.

Protocol: follow in this link

What we did in the lab:
Material:

Method:
1. Add all reagents in 1 ml Eppendorf

2. Digest during 2 h at 37 °C

3. For the reagent volumes, refer to the Table 9. Total volume = 50 µl

		C1
DNA (µl)	20	20
BamH I (µl)	1	1
Hind III (µl)	2	2
H₂0 (µl)	22	22
CutSmart(µl)	5	5

Table 9

5. Analysis and storage of the transformation done on June 6, 2016

6. Electrophoresis of pET43.1 and pSB1C3

7. Preculture of pET43.1a(+) DH5α to make stab culture

June 14, 2016:

8. Make a culture of DH5α pET43.1 to monitor the growth curve

June 15, 2016:

9. Make a growth curve of bacteria culture

10. Stab culture

11. Resuspension of C1 and C2 and all inserts synthesized by iDT

June 16, 2016:

12. Extraction of pET43.1a(+) and pSB1C3 DNA from agarose gel

13. Digestion of inserts (C1 and C2)

14. Dephosphorylation

15. Ligation of C1 and C2 with pET43.1a(+)

Team:Pasteur Paris/Microbiology week2

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Microbiology Notebook

Week 2

June 13, 2016: