Difference between revisions of "Team:Pasteur Paris/Microbiology week2"

*Table 3 : LB + CM34*
	Box 1	Box 2	Box 3
pET43.1a(+) DH5α	0	2	23
pET43.1a(+) DH5α (different batch)	Uncountable (Overgrown)	2	23
pET43.1a(+) DH5α (different batch)	Uncountable (Overgrown)	15	23

*Table 4 :*
	Box 1	Box 2
pSB1C3 DH5α	1	78
pSB1C3 DH5α (different batch)	38	Uncountable (Overgrown)

*Table 5*
	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 6*
	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

What we did on the lab:

Method:

1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 µl of carbenicillin 50 mg/ml added, swirl to mix.
2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total).
3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times.
4. Grow overnight at 37 °C and next day place it at 4°C.

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.

*Table 7*
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

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:

• 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:

*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

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 minute 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 minute and spin for 1 minute 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:

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.

*Table 9*
		C1
DNA (µl)	20	20
BamH I (µl)	1	1
Hind III (µl)	2	2
H₂0 (µl)	22	22
CutSmart(µl)	5	5

Aim:

Dephosphorylate our inserts

Protocol: follow in this link

Aim:

Resuspend C1, C2 and all inserts synthesized by iDT.

Protocol:

follow in this link

@@ Line 283: / Line 283: @@
        <td align = "center"; valign = "center">0</td>
        <td align = "center"; valign = "center">2</td>
+       <td align = "center"; valign = "center">23</td>
      </tr>
@@ Line 290: / Line 290: @@
        <td align = "center"; valign = "center">Uncountable (Overgrown)</td>
        <td align = "center"; valign = "center">2</td>
+      <td align = "center"; valign = "center">23</td>
      </tr>
      <tr>
@@ Line 295: / Line 296: @@
        <td align = "center"; valign = "center">Uncountable </br>(Overgrown)</td>
        <td align = "center"; valign = "center">15</td>
+      <td align = "center"; valign = "center">23</td>
      </tr>
 </tbody>

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(a+) 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(+)

Difference between revisions of "Team:Pasteur Paris/Microbiology week2"

Revision as of 21:30, 19 October 2016

click on the weeks

Microbiology Notebook

Week 2

June 13, 2016:

Difference between revisions of "Team:Pasteur Paris/Microbiology week2"

Revision as of 21:30, 19 October 2016

click on the weeks

Microbiology Notebook

Week 2

June 13, 2016:

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(a+) 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(+)

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