Difference between revisions of "Team:Pasteur Paris/Microbiology week5"

Aim:We do again the previous experiment to understand why transformation in our bacteria does not work.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+)a plamid (obtained with Midiprep done on June 8, 2016)
• enzyme restriction (XbaI / HindIII)
• Buffer Cutsmart 10X (NEB)
• H2O
• P10 pipet, P20 pipet, 1.5 mL eppendorf, 37°C and 65°C water bath

Method:
1. For our manipulation, we used XbaI and HindIII enzymes and we used Cutsmart 10 X buffer because it is the most appropriate buffer.
2. Follow the next table to volumes:

	pET43.1a(+) à 130 ng/µl (3 µg)
DNA (µl)	19
XbaI (µl)	2
Hind III (µl)	1
H20 (µl)	5
CutSmart 10X (µl)	24.5
TOTAL (µl)	51.5

Table 35

	= 260 nm	pET43.1a(+) XbaI/HindIII
N°1	A260/280	1.8
N°1	Cfinal	260 ng/µL
N°2	A260/A280	1.4
N°2	Cfinal	400 ng/µL

Table 36

Protocol: follow in this

Aim:Check if digestion was successfully done

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid (obtained with Midiprep on june 8, 2016)
• pET43.1a(+) plasmid digested by XbaI/HindIII
• gel 0.7% agarose
• TAE 0.5X buffer
• Electrophoresis generator ( at 50 V and after at 90 V)
• DNA ladder (Thermoscientific gene ruler 1kb)
• P10 and P20 pipet, 1.5 eppendorf,
electrophoresis BIORAD Mini-Sub Cell GT
Method:
1- Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts)
2- Fill electrophoresis chamber with TAE 1X
3- Follow the deposit Table 37:

Line	L1	L3	L5
Name	Marker weight	pET43.1a(+) X/H	pET43.1a(+) UNCUT
A_{DNA (µl)}	5	10	5
H20 (µl)	0	0	0
Load buffer 6X	0	2	1

Table 37

1- Put the voltmeter at 100 V for one hour
Results:

IMAGE Figure 7

We saw that in the line of the plasmid cut by XbaI and HindIII, we have two bands which don’t correspond to the uncut plasmid band. Moreover, The band of the plasmid cut by XbaI and HindIII have the appropriate weight. So we can cuclude that the digestion has worked. We can get back our plasmid cut with extraction gel.

Aim:To recover the pET43.1a(+) which has been digested, and to purify out the band from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:
• Results of electrophoresis (done previously)
• Gel extraction kit from Qiagen
• P10 and P20 pipet, 1.5 ml Eppendorf
Method:
To extract DNA we use the Gel Extraction Kit from Qiagen and we follow the different steps detailed in the kit.

• Eppendorf mass : 1.4043 g
• Eppendorf + Gel mass : 1.0817 g
• Gel mass : 1.4043 – 1.0817 = 322.6 mg

We must pour 3 volums of QG buffer, it means 967.8 µl.
We added 322 µl of isopropanol and we split in two equals volums our experiment

	DNA Masses (mg)	DNA Volumes (ml))
pET43.1a(+)	322.6	1.073

Table 38

Aim:After the electrophoresis, we saw that the digestion of pET43.1a(+) was done succesfully. Now we have to dephosphorylate it to avoid self-ligation.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid digested by XbaI/HindIII
• Dephosphorylation rSAP enzyme
• P10 and P200 pipet, 1.5 eppendorf, water bath (37 °C and 65 °C)
Method:
1. For volumes, refer on the next table :
We dephosphorylated 7.5*120 ng/µl, so 900 ng of pET43.1a(+)

DNA of pET43.1a(+)( µl)	rSAP ( µl)	Buffer 10X ( µl)	H2O ( µl)	TOTA ( µl)
7.5	2.58	6	43.92	60

Table 39 Results:

Aim:We want to obtain a second expression vector, this time with pET43.1 and C2. We do the same experiment as previously performed for C1.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylated
• C2 insert cut by XbaI/HindIII (done on the June,28 2016)
• T4 Ligase and Buffer 10X
• P10 and P200 pipet, 1.5 eppendorf, waterbath (37 °C and 65 °C)
Method:
1. For volumes, refer to the next table:
C2 = 9.2 ng/µl
We used 60 µl of pET43.1a(+) concentrated at 15 ng/µl (total 900 ng)

	1:1	1:3	Only pET43.1a(+)
pET43.1a(+) (50 ng)	6.7	6.7	6.7
Insert (C2) (µl)	1.74	5.22
T4 Ligase (µl)	1	1	1
Buffer 10X (µl)	1.5	1.5	1.5
H20 (µl)	4.06	0.58	5.8
TOTAL (µl)	15	15	15

Table 40

2. Incubate 10 min at room temperature, and put it at 65 °C during 10 min to inactivate the ligase. To keep it and use it after, we put it at -20 °C

Results:

Aim:

Protocol: follow in this link

What we did in the lab:
Materials:
Method:
Results:

@@ Line 579: / Line 579: @@
            <figcaption>
               <p>
-                 <U> Aim:</U></br></br>
+                 <U> Aim:</U>We want to obtain a second expression vector, this time with pET43.1 and C2. We do the same experiment as previously performed for C1.</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>
+                • pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylated</br>
+                • C2 insert cut by XbaI/HindIII (done on the June,28 2016)</br>
+                • T4 Ligase and Buffer 10X</br>
+                • P10 and P200 pipet, 1.5 eppendorf, waterbath (37 °C and 65 °C)</br>
                  <U>Method:</U></br>
+.  For volumes, refer to the next table:</br>
+                C2 = 9.2 ng/µl</br>
+                We used 60 µl of pET43.1a(+) concentrated at 15 ng/µl (total 900 ng)</br></br>
+                  <table>
+                    <thead>
+                      <tr>
+                        <th></th>
+                        <th>1:1</th>
+                        <th>1:3</th>
+                        <th>Only pET43.1a(+)</th>
+                      </tr>
+                    </thead>
+                    <tbody>
+                      <tr>
+                        <td><strong><p>pET43.1a(+) (50 ng)</p></strong></td>
+                        <td>6.7</td>
+                        <td>6.7</td>
+                        <td>6.7</td>
+                      </tr>
+                      <tr>
+                        <td><strong>Insert (C2) (µl)</strong></td>
+                        <td>1.74</td>
+                        <td>5.22</td>
+                        <td></td>
+                      </tr>
+                       <tr>
+                       <td><strong><p>T4 Ligase (µl)</p></strong></td>
+                        <td>1</td>
+                        <td>1</td>
+                        <td>1</td>
+                      </tr>
+                       <tr>
+                     <td><strong><p>Buffer 10X (µl)</p></strong></td>
+                        <td>1.5</td>
+                        <td>1.5</td>
+                        <td>1.5</td>
+                      </tr>
+                        <tr>
+                     <td><strong><p>H20 (µl)</p></strong></td>
+                        <td>4.06</td>
+                        <td>0.58</td>
+                        <td>5.8</td>
+                      </tr>
+                     <tr>
+                     <td><strong><p>TOTAL (µl)</p></strong></td>
+                        <td>15</td>
+                        <td>15</td>
+                        <td>15</td>
+                      </tr>
+                    </tbody>
+                  </table>
+                  <center>Table 40</center></br></br></br>
+.  Incubate 10 min at room temperature, and put it at 65 °C during 10 min to inactivate the ligase. To keep it and use it after, we put it at -20 °C</br></br>
                  <U>Results:</U></br></br>
              </p>

Difference between revisions of "Team:Pasteur Paris/Microbiology week5"

Revision as of 15:52, 15 October 2016

click on the weeks

Microbiology Notebook

Week 5

July 4, 2016:

47. Transformation of pSB1C3 and pET43.1a(+)

48. DNA measurements

49. Agarose gel (0.7 % agarose gel )

50. Electrophoresis on agarose gel of pET43.1a(+) digest by XbaI and HindIII

51. Extraction gel of pET43.1a(+) digest by XbaI and HindIII

52. Dephosphorylation of pET43.1a(+) (digest by XbaI/HindIII) done previously

July 5, 2016:

53. Dephosphorylation of pET43.1a(+)digest by XbaI/HindIII)

54. Ligation of dephosphorylated pET43.1a(+) with C2

55. Ligation of pET43.1a(+) dephosphorylate with C2

56. Electrophoresis on agarose gel of pET43.1 digest by XbaI and HindIII

57. Electrophoresis of pET43.1a(+) digested by XbaI and Hind III

58. Plasmid concentrations

59. Transformation of DH5 competent cells

July 6, 2016:

60. Verification of transformation of the 5/07/16

61. Cultivating colonies to recover the ligated plasmids-C1, or C2 inserts

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

63. Digestion of insert B2

64. ligation of insert B2 in pET43.1a(+)

July 7, 2016:

65. Check of the petri dish done on the July 6, 2016

66. Extraction of DNA of colonies from 06/07/2016

67. Agarose gel

68. Dosage of extracted DNA

69. Digestion of extracted DNA

70. Electrophoresis of pET43.1a(+)

71. Digestion of the recombinating plasmid

72. Electrophoresis of our results

July 8, 2016:

73. Transformation of bacteria BL21DE3