Aim

Prepare an 8% agarose gel for the electrophoresis of DNA samples.

Materials

• UltraPure™ Agarose (Invitrogen, 16500-100)
• UltraPure™ 10X TAE Buffer (Invitrogen, 15558)
• Gel Green Nucleic Acid Stain (Biotium, 41005)
• Scale
• Microwave
• Spatula
• Erlenmeyer (250 mL)
• Measuring cylinder
• PowerPac™ Basic (Bio-Rad, 1645050)
• Mini-Sub® Cell GT Horizontal Electrophoresis System (Bio-Rad, 1704406)

Procedure

1. Prepare 600 mL of TAE 1X by diluting 60mL of 10X buffer in 540mL of deionized water.
2. Weigh 0,6 g of agarose on a scale.
3. Place the agarose in an Erlenmeyer.
4. Fill the Erlenmeyer with 75 mL of TAE 1X.
5. Heat the Erlenmeyer for 2 min 30 s at 350W.
6. Mix and place it again in the microwave for an additional minute.
7. Let the mixture cool down a little bit and add 5 μL of Gel Green.
8. Pour the agarose in the horizontal electrophoresis system. Don’t forget to place the comb before!
9. Let the gel cool down for 20-30 minutes before deposing the samples.

Aim

Stock bacterial culture at -80 °C.

Materials

• Desired bacterial cultures on petri dish
• Sterile LB media
• Accurate antibiotics: Carbenicillin (50 mg/mL) or Chloramphenicol (25 mg/mL)
• Glycerol 50%
• Dry Ice
• Falcon 15 mL and 50 mL
• Erlenmeyer 125 mL
• Sterile cryotube
• Inoculation loop
• Pipette p200 + p20 and associated cones
• Plastic graduated pipette (25 mL)
• Electric Pipetman

Procedure

In advance:

• Prepare a stock solution of LB + desired antibiotic in 50 mL falcon tube depending on how many culture you would like to stock in glycerol.
• Prepare a sterile stock solution of glycerol 50 %.

1. In 15 ml sterile falcon, add 5 mL of LB media
2. Vortex the stock solution of antibiotic and add 5 µL to the LB
3. Using an inoculation loop, gently touch a colony of transformed bacteria from the petri dish, plastic side facing you.
4. Immerse and dip the inoculation loop in the liquid media and stir.
5. Place the liquid culture in the incubator at 37˚C and 180 rpm for 16 h.
6. After 16 h, centrifuge the tubes 5 minutes at 3000 rpm.
7. Discard supernatant.
8. Resuspend the pellet in 5 mL of LB.
9. Discard supernatant.
10. Resuspend the pellet in 1 mL of fresh sterile LB medium + desired antibiotic
11. In a 125 mL Erlenmeyer, add 1 mL of bacterial culture in 24 mL of LB + desired antibiotic.
12. Incubate the culture at 37°C and 180 rpm.
13. Measure the OD every hour for the first 3 h and then every 20 minutes.
14. When the OD reaches 0.6 to 0.7, withdraw 5 mL of the bacterial liquid culture and add 5 mL of glycerol 50%.
15. Vortex the tube.
16. Aliquot the 10 mL into sterile cryotubes.
17. Place into dry ice and freeze at -80°C.

Aim

Extract a specific DNA band from an agarose electrophoresis gel.

Materials

• QIAquick Gel Extraction Kit (Qiagen, 28706)
• Scale
• Scalpel
• Heating block
• Water bath

Procedure

According to the QIAquick Gel Extraction Kit's manual

1. Using a UV light, excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μL).
3. Incubate at 50°C for 10 minutes (or until the gel slice has completely dissolved). To help dissolve the gel, mix by vortexing the tube every 2 to 3 minutes during the incubation.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5. Add 1 gel volume of isopropanol to the sample and mix.
6. To bind DNA, pipet the sample onto the QIAquick column and apply vacuum. After the sample has passed through the column, switch off the vacuum source.
7. (Optional): Add 0.5 mL of Buffer QG to QIAquick column and apply vacuum.
8. To wash the column, add 0.75 mL of Buffer PE to QIAquick column and apply vacuum.
9. Transfer QIAquick column to a clean 1.5 mL microcentrifuge tube or to a provided 2ml collection tube. Centrifuge for 1 minutes at 13,000 rpm (~17,900 x g).
10. Place QIAquick column in a clean 1.5 mL microcentrifuge tube.
11. To elute DNA, add 50 μL of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the centre of the QIAquick membrane and centrifuge the column for 1 minute at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30 μL elution buffer, let stand for 1 min, and then centrifuge for 1 minute.

Aim

To perform the ligation of one or more inserts in a plasmid using the In-Fusion cloning kit.

Materials

• Stellar Competent cells (Takara Clontech)
• Linearized plasmid
• Purified insert(s)
• 5X In-Fusion HD Enzyme Premix (Takara Clontech)
• Control plasmid pUC19
• Control insert
• Deionized water
• Water bath at 50°C
• 1.5 mL Eppendorf tubes
• Heating block at 80°C
• Dry ice

Procedure

1. Set the mix between insert and linearized vector in molar ratio 2:1 and complete with distilled water to reach a reaction volume of 16 µL. The optimal quantity of vector is 100-150 ng.
2. Pre-heat vector and insert for 5 minutes at 80°C.
3. Put on ice for 3 minutes.
4. Add 4 µL 5X In-Fusion HD Enzyme Premix and let the cloning occur in a water bath at 50°C.
5. Set on ice and proceed to transformation in Stellar competent cells.

Aim

Materials

Procedure

Aim

Materials

Procedure