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

	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

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

	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

	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

Protocol: follow on this link

What we did on the lab:
Method:
1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 l 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 synthetized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer.
Add TE buffer in each tubes and refer to the next table for volumes

Name	Weight (mg)	Cfinal (ng/&micro ;l)	V(TE) (&micro ;l)
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. SpeI/ XbaI or BamHI/HindIII 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 (/ ~ 10min)
4. Load sample onto the column. Close the cap, spin for 1 min then discard flow though
5. Re insert column into collection tube. Add 200 µl 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:
Add all reagents in 1 ml Eppendorf

- Digest during 2 h at 37 °C
- For the reagent volumes, refer to the Table 9. Total volume = 50 µl

		C1
DNA (µl)	20	20
BamHI (µl)	1	1
HindIII (µl)	2	2
H20 (µl)	22	22
CutSmart(µl)	5	5

Table 9

Aim:

Protocol: follow in this link

@@ Line 257: / Line 257: @@
               <p>
               <U> Aim:</U> Check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br> Seed pET43.1 DH5&alpha; in LB + carbenicillin 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 combinations need to be stored beyond the lifetime of plate colonies. </br></br>
-              <U> Protocol:</U> follow in this link</br></br>
+              <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
               <U>What we did in the lab:</U></br>Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 &micro;g/ml (LB + CB50) or with chloramphenicol 34 &micro;g/ml (LB + CM34) were counted for the presence of individual distinct colonies. </br></br>LB + CB50</br></br>
@@ Line 446: / Line 446: @@
          <figcaption><p>
                <U> Aim:</U Repeat Midiprep for pET43.1a(+). </br>Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.</br></br>
-               <U> Protocol:</U> follow in this link</br></br>
+               <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
                <U>What we did in the lab:</U></br>
                <U>Materials:</U>
@@ Line 482: / Line 482: @@
              <p>
                 <U> Aim:</U> Use the preculture of June 13, 2016 to start the new culture.</br></br>
-                <U> Protocol:</U> follow in this link</br></br>
+                <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
                <U>What we did in the lab:</U></br>
                <U>Materials:</U></br>
@@ Line 516: / Line 516: @@
              <p>
                 <U> Aim:</U> to make a stab culture we need to capture the bacteria growing at the exponential phase </br> In order to do that we need to take optical density at 600 nm (OD600nm).</br></br>
-                <U> Protocol:</U> follow in this link</br></br>
+                <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
                <U>What we did in the lab:</U></br>
                <U>Materials:</U></br>
@@ Line 751: / Line 751: @@
   </br></br>
-              <U> Protocol:</U> follow in this link</br></br>
+              <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
               <U>What we did in the lab:</U></br>
               <U>Material:</U></br>
@@ Line 831: / Line 831: @@
               <U> Aim:</U> after digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2.  </br></br>
-              <U> Protocol:</U> follow in this link</br></br>
+              <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
               <U>What we did in the lab:</U></br>
               <U>Material:</U></br></br>
@@ Line 900: / Line 900: @@
               <U> Aim:</U></br></br>
-              <U> Protocol:</U> follow in this link</br></br>
+              <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
             </p>
@@ Line 914: / Line 914: @@
               <U> Aim:</U></br></br>
-              <U> Protocol:</U> follow in this link</br></br>
+              <U> Protocol:</U> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br>
             </p>

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(+)

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

Revision as of 15:45, 4 October 2016

click on the weeks

Microbiology Notebook

Week 2

June 13, 2016: