Team:Pasteur Paris/Microbiology week3

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Microbiology Notebook

Week 3

June 20, 2016:

Dosage of pET43.1

Checkin of stab cultures

Transformation of pET43.1a (+)

June 21, 2016:

Analyse

Dosage of pET43.1a(+)

Digestion of pET43.1 and C1 and C2

Electrophoresis of pET43.1a(+) and the inserts C1 and C2:

Gel extraction:

Dephosphorylation of pET43.1a(+):

June 22, 2016:

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

Transformation of pET43.1a(+) linked with C1 and C2

June 23, 2016:

Aim: On the June 6, 2016, we have done a transformation of pET43.1a(+) into DH5alpha and we dose it to know it concentration.

What we did in the lab:
Materials:
Method:
The culture for plasmid amplification was carried out in a large volume then aliquoted in several 1.5 ml Eppendorfs for centrifugation, since our Falcon size rotors do not go up high in G force.

We have nine 1 ml Eppendorf with 50L of pET43.1 in each tubes so we assemble all tubes in one.
1. Centrifuge the first Eppendorf and pour it into the second Eppendorf.
2. Do the same protocol until the last Eppendorf.
3. Use a plastic uvette (path length 1cm) to measure the DNA concentration.
4. Put in 1ml of TE1X with P1000, then take off 15L with P20 and add 15L of DNA with P20.
5. Analysis in spectrophotometer, Eppendorf biophotometer plus at 260 nm

Results:
C1=87 ng/L
C2 = 103.3 ng/L

Due to these bad results, we decided to use another spectrophotometer “Eppendorf Biophotometer”:
C1=0,2 ng/L
C2= 0,8 ng/L

Again, our results were not correct so we remeasure with utraspec 3100 pro from Amersham Bioscience.

Sample 1 Sample 2 Sample 3

A₂₆₀
0.043 0.59 0.037

A₂₈₀
0.026 0.01 0.016

A260 /A280
1.7 5.9 2.31

C_{dilué(ng/µl)}
2.15 1.95 1.85

C_{final(ng/µl)}
143.33 130 123.33

C_{medium(ng/µl)}
133.22 133.22 133.22

Table 10

We keep these results for the end of the experiment.
Then, we resuspend the insert by adding buffer TE1X in each tube (refer to the June 15, 2016 for the volumes).

Results: Colonies on the petri dish were counted. We saw bacterial colonies so the stab culture was successful.

Aim: Refer to June 16, 2016.

Protocol: follow in this link

What we did in the lab:
Refer to June 16, 2016, for protocol (dephosphorylation, ligation with C1 and C2 and transformation).
We spread the mix in petri dish with CB50 and CM34 to see if the transformation was successful.

Results: We analyse the transformation done yesterday. We don’t saw bacteria colonies on the dish, so transformations were not successful. To understand why bacterial colonies don’t grow up on petri dish, plasmid DNA is assayed.

Aim: To link pET43.1a(+) with C1 and C2 we assayed before plasmid DNA but perhaps our ligation doesn’t work. We want to know the concentration of our samples.

Protocol: follow in this link

What we did in the lab:
Materials:
Method:
1. We need more buffer solution so we dilute TE 10X by ten to have TE.
In a 250 ml burette, we had 25ml of TE and we complete with water until 250 ml. We aliquot the 250 ml in fixe 50 ml bottle.
2. We made a 15/1000 dilutions with our samples (15 L of DNA and 985L of water)
3. In a plastic uvette, we pour 1 ml of buffer TE, then we take back 15L and add of DNA
4. We analyse at 260 nm

>λ= 260 nm Sample 1 Sample 2

A₂₆₀

A₂₈₀

A260 /A280

C_{dilué(ng/µl)}
1.25 1

C_{final(ng/µl)}
250 200

C_{medium(ng/µl)}
225 225 225

Table 11

Aim: Our transformation do not work so we made a new digestion with BamHI and Hind III. This step will let us to do the transformation..
Protocol: follow in this link

What we did in the lab:
Materials:
Method:
1. Add all reagent in a 1 ml Eppendorf (1 for C1, 1 for C2 and the last one for pET43.1)
2. Let digest during 1h at 37°C then incubate 5 min at 65°C
For the reagent volumes, refer to the table.

pET43.1= 130 ng/L
BamHI= 20 000 U/ml

C1/C2= 10 ng/L
Hind III= 20 000 U/ml

Table 11

Aim: This step is needed to purify our DNA. Indeed, Multiclonal site (parts digested by enzymes but useless) cannot be removed with a PCR clean up because their molecular weight is too low.br>
Protocol: follow in this link

What we did in the lab:
Materials:
Method:
1. 1. Make an 0,7% agarose gel (refer to June 13, 2016)
2. Fill the electrophoresis chamber with TAEO,5X buffer
3. Follow the deposit table:

Voltmeter: Voltmètre PowerPac HC Biorad
V = 50V
Amp(T=0) = 0.01 A

pET43.1a(+) C1 C2

pET43.a(+)(ng/µl)
23 µl

C1 or C2 10 ng/µl (400 ng)
40 µl 40 µl

BamHI 20 000 U/ml (10 U)
0.5 µl 0.5 µl 0.5 µl

HindIII 20 000 U/ml (20 U)
1 µl 1 µl 1 V

Tampon
5 µl 5 µl 5 µl

TOTAL
50 µl 50 µl 50 µl

Table 12

Lines L1 L2 L3 L4 L5 L6 L7 L8-L9 L10 L11

Name
Molecular
weight pET43.a(+)
uncut pET43.1a(+)
B pET43.1a(+)
H pET43.1a(+)
B/H C1 C2

V(DNA) (µl) 15 15 15 15 15 15 15

V(H20) (µl) 0 0 0 0 0 0 0

V(Load buffer) (µl) 3 3 3 3 3 3 3

Table 13

Results:
Figure 3

Aim: We want to take back the plasmid and the inserts digested.
The electrophoresis has set up the waste and the parts needed
Protocol: follow in this link

What we did in the lab:
Materials:
Materials:
Method:
1. Cut agarose gel to have only parts of plasmid that we need and put it on an 1ml Eppendorf

Empty eppendorf (g) Eppendorf + gel (g) Final weight (mg)

m (pET43.1a(+)
0.99 1.12 127.2

m(C1) 0.99 1.07 76.5

m(C2) 0.99 1.09 106.1

Table 13

2. We use NEB extraction kit: De Macherey-Nagel_nucleospin-Gel and PCR Clean-up

For NT1 volumes: we need 200L of NT1 for 100 mg DNA
For pET43.1 volume = 254 L of NT1 buffer with p1000
For C1 volumes= 153L of NT1 buffer with p1000
For C2 volumes= 212L of NT1 buffer with p1000

Aim: After the digestion we dephosphorylate It to prevent that it binds without the insert.

Protocol: follow in this link

What we did in the lab:
Materials:
Materials: Method:
Refer to June 16, 2016
C(pET43.1) = 500 ng pour 20L
C(C1/C2) = 400 ng pour 20L

Aim: Let the pET43.1 to bind with the insert.

Protocol: follow in this link

What we did in the lab:
Materials:
Method:
1. Add all reagents in a 1ml Eppendorf for each sample. Follow the table. We have to do the ligation in the smallest volume to have more efficiency.S

1 :1
Lab Method 1 : 3
Lab Method Only Pet43.1a(+)
Lab Method 1 :1 Academic Method 1 :3
Academic Method

pET43.1a(+) (50 mg)
2 μl 2 μl 2 μl 2 μl 2 μl

Insert C1-C2
2.5 μl 7.5 μl 2.5 μl 7.5 μl

T4 ligase
1 μl 1 μl 1 μl 2 μl 2 μl

Tampon 10X
2 μl 2 μl 2 μl 2 μl 2 μl

H20
12.5 μl 8.5 μl 15 μl 12.3 μl 7.3 μl

20 μl 20 μl 20 μl 20 μl 20 μl

Table 15

2. Let ligate 10 min at 37°C then incubate 10 min at 65°C to inactivate the enzymes

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

Protocol: follow in this link

What we did in the lab:
Materials:
• Nanodrop (Thermofisher)
• Elution buffer from QIAGEN kit
• Microbiology equipment (Follow this link)

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

Results:

λ= 260 nm B1(1) B1(2) C2>th>

A_DNA
0.725 0.741 0.761

C final 36.3 ng/µl 37.0 ng/µl 38.0 ngµl

Absorbance

5. Preparation of the samples for sequencing: put 15 µl of DNA and add 2 µl of fitted primers
6. Send the samples for sequencing

Aim: : Enter the plasmid into the bacteria

Protocol: follow in this link

What we did in the lab:
refer to June 6, 2016 but with these quantities:
- L of bacteria
- L of DNA
- L of SOC

Spread each sample on a petri dish made of LB and CB50 (carbenicillin 50g/ml)

Aim: We want to assay DNA of C1 and C2 but we do not have enough insert so we tried different dilutions.
.
Protocol: follow in this link

What we did in the lab:
Method:
1. first, we use 1L of C1 and 49L of water
2. we made the blank with H20
3. we use the spectrophotometer utrospec 3100 pro_amersham Bioscience at =260 nm
4. the results were not convincing so we decided to use a Nanodrop
Results:

>λ= 260 nm C1 C2

A260 /A280
1.71 1.8

A260 /A230
0.39 0.39

Cfinal
9.7 ng/μl 11 ng/μl

Table 16