Team:Pasteur Paris/Microbiology week15

Aim: To have the right restriction sites to perform the ligation and the cloning.
We choose appropriate restriction sites based on our inserts.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Spe I (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer
• pSB1C3 (48ng/µl)

Method:
1. Mix all the reagents and let digest during 1 hour at 37°C
⚠ Big volumes must be added first!

*Table 143*
Reactants	pSB1C3
Vol_DNA	25 µl
Vol_XbaI	10 µl
Vol_SpeI	10 µl
Vol_{buffer (10X) (Cutsmart)}	5 µl
Vol_total	50 µl

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Add 2 µl of rSAP in order to perform the dephosphorylation.
3. Store at -20°C

Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher)

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

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

Results:

*Table 144 : Absorbance*
Absorbance at 260nm	A260/280	Concentration (ng/µl )
pSB1C3 (1)	1.94	115.7
pSB1C3 (2)	1.93	13.0
B1-col6	1.88	393.3
B1-col8_{(diluted 1/2)}	1.93	74.1
B2-col7	1.89	84.8
B2-col9	1.90	307.1

Aim: To have the right restriction sites to perform the ligation and the cloning.
We choose appropriate restriction sites based on our inserts.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Spe I (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37°C water bath
• UV spectrophotometer
• pSB1C3 (48ng/µl)

Method:
Mix all the reagents and let digest during 1 hour at 37°C

⚠ Big volumes must be added first!

*Table 145*
Reactants	Each sample	Global mix
Vol_DNA	25 µl	0 µl
Vol_XbaI	1 µl	18 µl
Vol_SpeI	0.5 µl	9 µl
Vol_{buffer 2.1}	5 µl	90 µl
Vol_H2O	18.5 µl	333 µl
Vol_total	50 µl	450 µl

2. Incubate 10 min at 65°C to inactivate the enzymes.
3. Add 2 µl of rSAP in order to perform the dephosphorylation.
3. Store at -20°C

Aim: This step check the digestion efficiency of B1 (2 tubes)/B2 (2 tubes)/ C1 / C2 / A1 / A2 / D2 / E1 / E2.
Moreover, the inserts will be purified during this step because they will be extracted from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:
• Electrophoresis cuve
• TAE 1X
• Gene ruler (Thermoscientific 1kb plus)
• Loading dye
• Agarose
• UV table
• Ethidium bromide drops (EB)

Method:
Each well will contain 25 µl of DNA and 6 µl of Loading Dye. Follow the next deposit table :

Line 1:
Ladder (6 µl) | Ø | B2-col7 | Ø | B2-col9 | Ø | B1-col6 | Ø | B1-col9 | Ø | C1 | Ø | C2

Line 2:
Ladder (6 µl) | Ø | D2 | Ø | E1 | Ø | E2 | Ø | pSB1C€3 | Ø | A1 | Ø | A2

Aim: To perform a gel extraction to isolate insert DNA purified from its plasmid thanks to the migration. We use the gel made before with inserts B1 (2 tubes)/B2 (2 tubes)/ C1 / C2 / A1 / A2 / D2 / E1 / E2 but we only extract B2 bands.

Protocol: follow in this link

What we did in the lab:
Materials:
• Scalpel
• 2 ml Eppendorfs
• Balance
• UV table
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link) • QIAGEN Gel Extraction Kit

Method:
⚠ Be aware of the risks! UV light burns the eyes and skin so make sure you have the right protection
Follow QIAGEN Kit steps according to the next tables for the volumes of QG buffer.

*Table 146 : Masses*
Insert	Mass of gel (mg)	Volume of QG Buffer (µl)
B2-col7	190	570
B2-col9	170	510
B1-col6	80	240
B1-col8	140	420
C1	150	450
C2	130	390
D2	150	450
E1	150	450
E2	180	540
pSB1C3	210	630
A1	170	510
A2	210	630

Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher)
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 1ul of your sample on the Nanodrop
5. Make the measure and clean the Nanodrop between each measure

Results:

*Table 147 : Absorbance*
Absorbance at 260 nm	A260/280	Concentration (ng/µl)
B2-col7	2.12	3.7
B2-col9	2.19	5.3
B1-col6	2.04	3.2
B1-col8	1.98	3.7
C1	1.78	5.0
C2	1.90	307.1
D2	1.84	5.2
E1	2.30	3.9
E2	2.17	3.8
pSB1C3	1.98	13.1
A1	2.11	4.8
A2	2.45	4.3

Team:Pasteur Paris/Microbiology week15

click on the weeks

Microbiology Notebook

Week 15

September 12, 2016:

242. Digestion of the plasmid pSB1C3

September 15, 2016:

243. Measure the amount of DNA extracted from the miniprep

244. Digestion of the plasmid pSB1C3 and inserts A1/A2/B1/B2/C1/C2/D1/D2/E1/E2

245. Electrophoresis on agarose gel

246. DNA extraction

September 16, 2016:

247. Measure the amount of DNA extracted from the miniprep