PCR

We used the Thermo Scientific Phusion High-Fidelity DNA Polymerase. Amplification of templates with high GC content, high secondary structure, low template concentrations or long amplicons may require further optimization.

Materials

• PCR thermocycler
• PCR tubes
• nuclease-free water
• dNTP
• Phusion HF Buffer (X5) or GC Buffer
• Primers (both forward and reverse)
• Template DNA
• Phusion polymerase

Procedure

All components should be mixed and centrifuged prior to use. It is important to add Phusion DNA Polymerase last in order to prevent any primer degradation caused by the 3´→ 5´ exonuclease activity.

Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors.

Use of high quality, purified DNA templates greatly enhances the success of PCR.

1. We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).

   Component	50 μL	final concentration
   Nuclease-free water	qs 50 μL
   Buffer Phusion HF (5X)	10μL	1X
   10 mM dNTPs	1 μL	200 μM
   10 μM Forward primer	2.5 μL	0.5 μM
   10 μM Reverse Primer	2.5 μL	0.5 μM
   DNA template (10 ng/μL)	1 μL	10ng
   Phusion DNA Polymerase	0.5 μL	1.0 U/0.5 μL of reaction

   Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary
2. Transfer PCR tubes from ice to a PCR machine with the block preheated to 98°C and begin thermocycling:

   Step	Temperature	Time
   Initial denaturation	98°C	45 sec
   30 cycles	98°C	15 sec
   	55°C	30 sec
   	72°C	30 sec/kb
   Final extension	72°C	5 min
   Hold	4°C	hold

   Parts	Length	Time of extension
   pGAP-cOT2 / pGAP-DNY15 / pGAP-Leucro / YFP / DsRed	1 kb	30 sec
   harveyi 1 / 2 / 3 / Vc and Vh	2 kb	60 sec
   Odr10-cOT2	3 kb	90 sec

3. Then purify the products thanks to PCR purification kit

PCR purification

Introduction

This protocol was extracted from Invitrogen PureLink® PCR Purification Kit. Refer to this protocol for troubleshooting. Use the PureLink® PCR Purification Kit to efficiently remove primers, dNTPs, enzymes, and salts from PCR products in less than 15 minutes. Use the kit with Binding Buffer High-Cutoff (B3) to remove primer dimers or short spurious PCR products. The purified PCR product is suitable for automated fluorescent DNA sequencing, restriction enzyme digestion, and cloning.

Materials

• Binding Buffer (B2)
• Binding Buffer High-Cutoff (B3)
• Wash Buffer (W1)
• Elution Buffer; 10 mM Tris-HCl, pH 8.5 (E1)
• PureLink® PCR Spin Columns with Collection Tubes
• PureLink® Elution Tubes (1.7 mL)
• 50–100 μL PCR product
• 100% isopropanol
• 96–100% ethanol
• Sterile, distilled water (pH>7.0)
• Microcentrifuge capable of achieving >10,000 × g

Procedure

/!\ The PureLink® PCR Purification Kit buffers contain guanidine hydrochloride and isopropanol. Always wear a laboratory coat, disposable gloves, and eye protection when handling buffers.

/!\ Do not add bleach or acidic solutions directly to solutions containing guanidine hydrochloride or sample preparation waste because it forms reactive compounds and toxic gases when mixed with bleach or acids.

Follow the recommendations below to obtain the best results:

• Maintain a PCR volume of 50–100 μL
• Save an aliquot of PCR products before purification to verify and check the amplicon on the gel
• Use a centrifuge at room temperature for all steps
• Pipet the Elution Buffer (E1) in the center of the column and perform a 1 minute incubation
• Always use sterile water with pH 7–8.5, if you are using water for elution

1. Before starting. Add isopropanol to the Binding Buffers and ethanol to the Wash Buffer according to the following table. After adding isopropanol or ethanol, store all buffers at room temperature.

   Buffer	Cat. no. K3100-01
   Binding Buffer (B2)	10mL 100% isopropranol
   Binding Buffer HC (B3)	2.3mL 100% isopropranol
   Wash Bufer (W1)	64mL 96-100% isopropranol

2. Binding DNA.
3. Add 4 volumes of PureLink® Binding Buffer (B2) with isopropanol (see before starting) or Binding Buffer HC (B3) with isopropanol (see before starting) to 1 volume of the PCR product (50–100 μL). Mix well.
4. Remove a PureLink® Spin Column in a Collection Tube from the package.
5. Add the sample with the appropriate Binding Buffer (from step 1 of this procedure) to the PureLink® Spin Column.
6. Centrifuge the column at room temperature at 10,000 × g for 1 minute.
7. Discard the flow through and place the spin column into the collection tube.
8. Washing DNA
9. Add 650 μL of Wash Buffer with ethanol (see before starting) to the column.
10. Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube.
11. Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube. Then let the residual ethanol evaporate by placing the open column on the collection tube and let it sit for 5 mins.
12. Eluting DNA.
13. Place the spin column in a clean 1.7-mL PureLink® Elution Tube supplied with the kit.
14. Add 30 μL of Elution Buffer (10 mM Tris-HCl, pH 8.5) or sterile, distilled water (pH >7.0) to the center of the column.
15. Incubate the column at room temperature for 1 minute.
16. Centrifuge the column at maximum speed for 2 minutes.
17. The elution tube contains the purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μL. Store the purified PCR product at –20°C or use the PCR product for the desired downstream application.

Colony PCR

Introduction

This protocol was elaborated thanks to the help of Anthony Henras.

Materials

10 μL of 0.02N NaOH / 1 PCR

Procedure

1. Resuspend the equivalent of the tip of a P1000 pipette of the colony in 10 μL of 0.02N NaOH
2. Mix well (vortex)
3. Incubate 5 min at 95°C and then chill on ice for 10 min at 4°C (program the thermocycler to do so (Program YeastLysis))
4. For each PCR mix:
   NOTE: mix on ice and put on the thermocycler directly after mixing

   Component	Volume (μL)
   Previous cell extract	2
   Taq Pol Buffer	10
   Forward oligo 100 10 μM	0.5
   Reverse oligo 100 10 μM	0.5
   dNTP	1
   H2O	35.6
   Taq DNA polymerase	0.4

5. Put on a thermocycler and start this cycle:

   	95°C	5 min
   35 cycles	95°C	30 sec
   	55°C	1 min
   	72°C	3 min
   	72°C	10 min
   	22°C	∞

6. Migration on gel to check the results

Gel extraction of DNA

Procedure

Please, before doing your preparative gel, use one sample to make an analityc one !

1. Equilibrate a water bath or heat block to 50°C.
2. Excise a minimal area of gel containing the DNA fragment of interest.
   
   • Crucial: To protect the UV box, it is a good idea to place the gel on a glass plate if available.
   • Try to get as little excess gel around the band as possible.
3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.
4. Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:

   Gel	Tube	Buffer L3 Volume
   ≤2% agarose	1.7 mL polypropylene	3:1 (i.e., 1.2 mL Buffer L3: 400 mg gel piece)
   >2% agarose	5 mL polypropylene	6:1 (i.e., 2.4 mL Buffer L3: 400 mg gel piece)

5. Place the tube with the gel slice and Buffer L3 into a 50°C water bath or heat block. Incubate the tube at 50°C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution.
   • Note: High concentration gels (>2% agarose) or large gel slices may take longer than 10 minutes to dissolve.
6. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes.
   • Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
7. Before Starting: Add ethanol to the Wash Buffer (W1) according to the label on the bottle.
8. Purifying DNA Using a Centrifuge
9. Load. Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel.
   • Note: The column reservoir capacity is 850 μL.
10. Bind. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
11. Wash. Add 500 μL Wash Buffer (W1) containing ethanol to the column.
12. Remove Buffer. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
13. Remove Ethanol. Centrifuge the column at maximum speed for 1–2 minutes. Discard the flow-through.
14. Elute. Place the column into a Recovery Tube. Add 30 μL Elution Buffer (E5) to the center of the column. Incubate the tube for 1 minute at room temperature.
15. Collect. Centrifuge the tube at >12,000 × g for 1 minute.
16. Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at −20°C for long-term storage.

Migration on agarose gel.

Introduction

This protocol is the classical one used for electrophoresis. - You can adapt the concentration of agar according to the length of your fragment 1% agar if the DNA fragments are big 2% agar if the DNA fragments are small (the bigger fragment are sticked together) - Adapt the volume of the gel 15 to 30 mL for small gels and 150 to 200 mL for big gels

Procedure

1. Thoroughly rinse gel housing and well-comb with dH2O.
2. Place gel mold perpendicular to flow direction, ensuring proper sealing of rubber gaskets.
3. Add the calculated amounts of 0.5xTBE and agarose to a fresh Erlenmeyer flask.
4. Heat in microwave until mixture can be dissolved.
   • CRITICAL: Do not let the mixture boil over and out of the flask. Typical heating time for 50mL in a 2.45GHz microwave oven at full power is 30s. USE HEAT GLOVES
5. Gently swirl until well mixed and gently swirling periodically until ~55°C.
6. Gently pour molten agarose gel into housing, avoiding air bubbles.
7. Place desired well comb in desired position.
8. Once gelled, carefully remove well comb in a uniform fashion.
9. Remove gel mold and place in parallel direction to flow
   • CRITICAL: the deposit line has to be at the anode (negative pole)
10. Fill gel box with 0.5 xTAE until the gel is well covered.
11. Place the ladder on the gel, the native and digested plasmid (write down the gel map)
    • TIP: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip.
12. Run the electrophoresis for 20-30min at 100V until the dye line is approximately 80% of the way down the gel
13. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
14. Place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, r
15. Place the gel into a container filled with water and destain for 5 mins.
16. Reaveal under UV lamp, visualize your DNA fragments

Miniprep.

Introduction

This protocol was taken from the ThermoScientific GeneJET Plasmid Miniprep Kit. Safety: Both the Lysis Solution and the Neutralization Solution contain irritants. Wear gloves when handling these solutions.

Procedure

• Note: All steps should be carried out at room temperature. All centrifugations should be carried out in a microcentrifuge at ≥ 12 000 x g (10 000-14 000 rpm, depending on the rotor type).
• Be sure that the concentrated solutions have been diluted with the appropriated buffer

1. Pick a single colony from a freshly streaked selective plate to inoculate 5mL of LB medium supplemented with the appropriate selection antibiotic.
2. Incubate for 12-16 hours at 37°C while shaking at 200-250 rpm
3. Centrifugate the bacterial culture, >12 000 g in a microcentrifuge for 2 minutes at room temperature. Repeat until there is no more media.
4. Add to the pelleted cells:
   • 250 μL of Resuspension Solution and vortex
   • 250 μL of Lysis Solution and invert the tube 4-6 times. WAIT 2 min
   • 350 μL of Neutralization Solution and invert the tube 4-6 times.
   • Lysis buffer must be neutralized before 5 minutes
5. Centrifuge 5 minutes.
6. Transfer the supernatant to the Thermo Scientific GeneJET Spin Column. Centrifuge 1 minute
7. Add 500 μL of Wash Solution and centrifuge for 60 s and discard the flow-through
8. Repeat step 5.
9. Centrifuge empty column for 1 minute.
10. Dry for 5 minutes
11. Transfer the column into a new tube.
12. Add 30 μL of Elution Buffer to the column and incubate 2 minutes.
13. Centrifuge 2 minutes.
14. Collect the flow-through.

Ligation

Introduction

Please see the NEB website for supporting information on this protocol.

Materials

• 10X T4 DNA Ligase Reaction Buffer
• T4 DNA Ligase
• Vector DNA (4kb)
• Insert DNA (1kb)
• nuclease-free water

Procedure

Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEB calculator to calculate molar ratios.

1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature. Tip: Alicuote the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.
2. Set up the following reaction in a microcentrifuge tube on ice:

   Component	Volume (µL)
   10X T4 DNA Ligase Buffer	2
   Vector DNA: 50 ng (0.020 pmol)
   Insert DNA: 37.5 ng (0.060 pmol)
   Nuclease-free water	17
   T4 DNA Ligase	1
   Total	20

3. Gently mix the reaction by pipetting up and down and microfuge briefly.
4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.
5. Heat inactivate at 65 degrees C for 10 minutes.
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Use 25 uL DH5α cells, and add 2 uL of reaction mixture.

Chemical transformation (RbCl method)

Introduction

This protocol was given by Stéphanie. The aim is to make yourself Top10 competent cells.

Materials

• 2 * Steri cup 250mL
• TrisEDTA

Procedure

Media and Solutions

1. 500 mL LB
2. 200 mL TFB1:
   • 0.59 g KOAc (Cf=30 mM)
   • 2.42 g RbCl (100 mM)
   • 0.29 g CaCl2 2H2O (10 mM)
   • 1.98 g MnCl2 4H2O (50 mM)
   • 30 g Glycerol (15% wt/vol)
   • Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH). Add dH2O to 200 mL. Filter sterilize. Store refrigerated at 4°C.
3. 200 mL TFB2:
   • 0.42 g MOPS (10 mM)
   • 2.21 g CaCl2 2H2O
   • 0.24 g RbCl (10 mM)
   • 30 g Glycerol (15% wt/vol)
   • Adjust to pH 6.5 with KOH. Add dH2O to 200 mL. Filter sterilize. Store refrigerated at 4°C.

Preparation of Competent Cells

4. Streak cells from frozen stock onto LB plate. Incubate O/N at 37°C.
5. Pick a single fresh colony to inoculate 5 mL of LB medium. Grow O/N at 37°C. Do not vortex cells at any time after this point in the procedure.
6. Dilute 1 mL of culture into 50 mL LB medium prewarmed to 37°C. Grow at 37°C for 2 hours with agitation. Volumes can be scaled up 5X and all of the 5 mL overnight culture can be used.
7. Transfer culture to sterile 50 mL tube. Chill on ice 10-15 minutes.
8. Centrifuge for 10 mintutes at 2000 rpm at 4°C. Immediately aspirate off all the supernatant. Do not allow cells to warm above 4°C at any time in this procedure.
9. Resuspend cells in 10 mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass of plastic pipette.
10. Incubate cells on ice for 5 minutes.
11. Repeat step 8
12. Resuspend cells in 2 mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic).
13. Incubate cells on ice for 15 minutes. Cells may be used for transformation or frozen. To freeze: aliquot cells 100 µL volumes into prechilled 0.5 mL microcentrifuge tubes (on ice). Freeze immediately on dry ice. Stire cells frozen at -80°C.

Transformation of competent cells

14. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed (i.e., at ~0°C). Then, immediately place on ice for about 5 minutes.
15. Set up transformation as follows:
    Add to 15 mL plastic round bottom tube on ice:
    • 0-9 µL TE (Tris 10mM + EDTA 1mM)
    • 1-10 µL DNA (10-100 ng)
    • 10 µL final volume → /!\ 10% max of the cell competent volume
16. Add 100 µL of competent cells and mix by gentle repipetting. This method can be scaled down 2- to 4-fold. The maximum volume of DNA should be ~1/10 volume of cells and the maximum mass should be <= 100 ng of DNA for 100 µL of cells.
17. Incubate cells on ice for 20-30 minutes.
18. Heat shock the cells exactly 90 seconds at 42°C.
19. Return cells on ice 2 minutes.
20. Add 1 mL of LB medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking. For blue/white color selection, spread IPTG and X-gal on plates now and hold at 37°C until use
21. Plate 0.1 - 0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic (adn IPTG and X-gal if needed). Incubate overnight at 37°C. Place at 4°C to store and/or enhance blue color. Note: The next day, liquid cultures of the transformants can be left 8 hours before the miniprep. In the best-case scenario, do the liquid culture at 8am and do the miniprep at 4pm.

Testing competent cells

22. Transform 100 µL of cells with 1 µL (10 pg) of pUC19 monomer (0.01 µg/µL).
23. Plate 0.25 mL of transformation mixture. Incubate overnight at 37°C.
24. Count CFU and calculate efficiency. Efficiency =# of colonies per µg =# of colonies X4 X 105. You should obtain 1-5 X 10⁷/µg from competent cells after one freeze-thaw cycle.

Enzymatic digestion of DNA.

Introduction

This protocol was extracted from the protocol from NEB website.

Materials

• For analysis digestion:
  • Eppendorfs
  • 1 μg of DNA
  • 1 μL 10X buffer (most enzymes can be used in Cutsmart buffer, check on NEB website)
  • 1U enzyme pour 1 μg ADN (0,5 μL for 1 μg DNA)
  • H₂O qsp 10 μL
  • heating plate
• For preparative digestion:
  • Keep the same proportions and scale up for 30µL of DNA on 100µL final
• If cut by the same Enzyme, please prepare a MIX with n+1 (n = number of sample)
• For gel migration, add 2 μL of loading dye for each 10 μL mix

Procedure

1. Mix all the elements
2. Incubate 1h at enzyme specific temperature (usually 37°C)
3. Check if heat inactivation is required and do it accordingly /!\ if inactivation is done at high temperature put on ice after inactivation and then centrifuge to keep the evaporated water.

Electroporation of P.pastoris.

Introduction

Protocol from Lin-Cereghino, J., Wong, W., Xiong, S., Giang, W., Luong, L., Vu, J., Johnson, S. and Lin-Cereghino, G. (2005). Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. BioTechniques, 38(1), pp.44-48.

Materials

• ice
• linearized plasmid (with AvrII)
• competent cells from the protocol cell preparation
• electroporation apparatus
• 1.0M sorbitol
• YPD
• plates with gradient of zeocin

Procedure

1. Mix approximately 4-8μL (50–100 ng) of dialysed linearized plasmid DNA with 40 μL of competent cells in an electroporation cuvette
2. Incubate for 2 min on ice
3. Pulse 1500V, 25μF, 200Ω (You should have a Ꞇ between 4 and 5 ms. If it is >5ms, there were too many ions in the mix. It can kill cells.) (was done previously with 1500V, 10μF, 600Ω -> worked)
4. Resuspend immediately samples in 0.5 mL 1.0 M sorbitol and 0.5 mL YPD, incubate in a 30°C shaker for 1h30, and then plate on media containing increasing concentrations of zeocin (100, 250, 500, or 1000 μg/mL) for the selection of multicop