Difference between revisions of "Team:Pasteur Paris/Microbiology week13"

Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2)

What we did in the lab:
Materials:
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
1. Analyze absorbance at 260nm
2. Clean the Nanodrop with water
3. Make the blank with 1µl of elution buffer
4. Put 1 µl of your sample on the Nanodrop
5. Make the measure and clean the Nanodrop between each measure

Results:

*Table 1 : Absorbance*
Absorbance at 260nm	A₂₆₀	A₂₈₀	A_260/280	Concentration (ng/µl)
A1(1)	0.202	0.124	1.62	10.1
A1(2)	0.259	0.169	1.53	13.0
A2(1)	0.179	0.105	1.70	8.9
A2(2)	0.179	0.103	1.73	8.9
A2(3)	0.098	0.052	1.86	4.9
A2(4)	0.152	0.099	1.53	7.6

Aim: To start a culture for Miniprep of insert C2 in pET43.1a in BL21(DE3) from 22/08 and A1/C2 in pET43.1a in TOP1O.
In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 50 ml erlenmeyers
• Carbenicillin 50 mg/ml
• LB medium

Method:
1. One colony is picked from the plates and shaken in 25.0 ml of LB supplemented with Carbenicillin at 50 µg/ml.
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.

Aim: Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reaches an optical density of 0.7.

Protocol: follow in this link

What we did in the lab:
Materials:
• Two 2 l erlenmeyers
• LB (Luria broth)
• IPTG (0.1 M)
• Precultures in 25 ml Erlenmeyers
• UV spectrophotometer (Ultrospec 3100)
• Shaking incubator (INFORS HT)
• Centrifuge
• Buffer A (50mM Tris, 150mM of NaCl)

Method:
1. Put 1 l of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150 rpm.
2. Once warmed, add 12.5 ml of preculture in each erlenmeyer.
3. Let grow in the shaking incubator.
4. Measure the absorbance with the UV spectrophotometer every 30min

*Table 140 : Optical Density*
Time	C2 (1)	C2 (2)
9:36_AM	0.024	0.023
10:06_AM	0.049	0.046
11:02_AM	0.175	0.165
11:36_AM	0.395	0.402
12:05_AM	0.568	0.540
12:27_AM	0.658	0.638
12:38_AM	0.694	0.680
14:31_PM	0.872	0.859

5. Add IPTG to reach a concentration of 0.1mM. (12:57_AM)
6. The last measure before induction is pelleted 3min at 8000g and stored at -20°C.
  7. Let induce overnight in the shaking incubator
8. After 7 hours of induction, measure the OD_{600 nm} and store the measure pelleted at -20°C.
9. Centrifuge at 4500RPM the culture and throw out the supernatant, the pellet resuspended in 5ml of buffer A are stored at -80°C before protein extraction.

Aim: To perform a Midiprep to isolate plasmid DNA of pET43.1(a+) with the inserts A1/C2 from cultures made on the 1^set of August.
The amplification method to increase the amount of plasmid is called Midiprep.

Protocol: follow in this link

What we did in the lab:
Materials:
• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• QIAGEN Miniprep kit
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below.
Follow QIAGEN kit steps with a final volume of 100 µl

@@ Line 347: / Line 347: @@
                    <U>Method:</U><br/>
-                   One colony is picked from the plates and shaken in 25.0 ml of LB supplemented with Carbenicillin at 50 &#181;g/ml.<br/>
+. One colony is picked from the plates and shaken in 25.0 ml of LB supplemented with Carbenicillin at 50 &#181;g/ml.<br/>
-                   The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.<br/>
+. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.<br/>
                    <br/><br/><br/>
@@ Line 367: / Line 367: @@
                    <U>Materials:</U><br/>
                    &bull; Two 2 l erlenmeyers<br/>
-                   &bull; LB (lysogeny Luria broth)<br/>
+                   &bull; LB (Luria broth)<br/>
                    &bull; IPTG (0.1 M)<br/>
-                   &bull; Precultures in 25 ml erlenmeyers<br/>
+                   &bull; Precultures in 25 ml Erlenmeyers<br/>
                    &bull; UV spectrophotometer (Ultrospec 3100)<br/>
                    &bull; Shaking incubator (INFORS HT)<br/>
@@ Line 382: / Line 382: @@
                    <table>
-                    <caption align="bottom" align="center"><i><p>Table 1 : Optical Density</p></i></caption>
+                    <caption align="bottom" align="center"><i><p><U>Table 140 :</U> Optical Density</p></i></caption>
                      <thead>
                           <tr>
@@ Line 437: / Line 437: @@
 . The last measure before induction is pelleted 3min at 8000g and stored at -20°C.<br/>
 . Let induce overnight in the shaking incubator<br/>
-. After 7 hours of induction, measure the OD and store the measure pelleted at -20°C.<br/>
+. After 7 hours of induction, measure the OD<sub>600 nm</sub> and store the measure pelleted at -20°C.<br/>
 . Centrifuge at 4500RPM the culture and throw out the supernatant, the pellet resuspended in 5ml of buffer A are stored at -80°C before protein extraction.
                      <br/><br/><br/>

Difference between revisions of "Team:Pasteur Paris/Microbiology week13"

Latest revision as of 03:38, 20 October 2016

click on the weeks

Microbiology Notebook

Week 13

August 29, 2016:

232. Measure the amount of DNA extracted from the miniprep

September 1, 2016:

233. Harvest the culture with Midiprep

September 2, 2016:

234. Growth of bacteria

235. Extraction of plasmid DNA