Team:USP-Brazil/Protocols

Protocols

Cloning

---

Plasmid DNA isolation

Extraction of plasmid DNA from E. coli by Alkaline Lysis – Miniprep

1.1) Formation of pellets^*

• Transfer 1.5 ml of E. coli sample to eppendorf.
• Centrifuge at 14000 RPM for one minute at 4°C.
• Discard the supernatant.
• Add the remaining E. coli, centrifuge again and discard the supernatant.
• Resuspend in 200 μL of Solution 1 (P1) and vortex to dilute the entire pellet.

^*: Keep all on ice

1.2) Extraction of DNA

• Add 200 μL of Solution 2 (P2) freshly made.

Solution 2^*:
For 2 mL: 1860 μL of milliQ water, 40 μL of NaOH 10N, and 100 μL of SDS 20%.
For 5 mL: 4650 μL of milliQ water, 100 μL of NaOH 10N, and 250 μL of SDS 20%.
^*: The reagents MUST be added in the order described. Stir after each mix. Do not put ice or you might have to redo if a precipitate (crystal) forms.

• Gently invert 6 times.
• Leave at room temperature for 5 minutes.
• Add 200 μL of Solution 3 (P3), flip and put on ice immediately.
• Centrifuge at 14000 RPM for 20 minutes at 4°C.
• Collect the supernatant (with pipette) without getting any of the precipitate pellet moved into the new eppendorf. Collect 200 μL at a time with the pipette tip to prevent getting any of the pellet.

1.3) Precipitation of DNA

• Add 400 μL of isopropanol. Invert 5 times.
• Connect the speed vaccum.
• Centrifuge at 14000 RPM for 10 minutes at 4°C.
• Discard the supernatant and invert eppendorf over paper towel.

1.4) Dry DNA

• Add 500 μL of ethanol 70%. Invert 5 times.
• Centrifuge at 14000 RPM for 5 minutes at 4°C.
• Discard and speed vacuum for 15 minutes.

1.5) Resuspend the DNA and degrade the RNA

• Resuspend in 30 μL of milliQ water with light finger touches.
• Treat with 5 μL RNAse per sample. Resuspend with light finger touches.
• Leave at 37°C for at least half an hour.
• Place in 20°C freezer for overnight storage.

2) Quantification by Nanodrop

• Connect software.
• Select option “Nucleic Acid.”
• Place 2 μL of milliQ water on each pedestal and clean with paper towel.
• Place 2 μL of milliQ water to calibrate the instrument and press “OK.”
• Carry out the “Blank” using 2 μL of either milliQ water or buffer, depending on what you used to dilute your DNA sample.
• Place 2 μL of sample and click measure.
• Observe the quantification of DNA in ng/μL.
• Also observe the ratio of 260/280, which must be around or greater than 1.7-1.8.

3) Digest to linearize the plasmid

Reagents	1 reaction
ECO RI*	1μL
Enzyme Buffer	2μL
DNA	2μL
H2O	15μL
Total	20μL

* place the enzyme last

Solutions P1, P2 and P3

P1	P2	P3
23 ml glucose 20%	H2O 5580 ul	KaOH 3M
GTE 500 ml	NaOH 120 ul	60 ml KOAC 5M
10 ml EDTA 0.5 M pH 8,0	300 ul SDS 20%	11.5 ml Glacial Acetic Acid
12.5 ml of Tris HCl 1M pH 7,0		Water to 100 ml
Water to 500 ml		11.5 ml Glacial Acetic Acid
Autoclave before using		Autoclave before using

---

Protocol for Miniprep from Plate (used when there was a large amount of samples)

• Take the plates out of the fridge
• Add 1 µL of media (LB) with ampicillin (100 µg/mL) to each well
• Choose colonies by poking with a sterile toothpick and inoculate (put toothpick in well). Cover the 96-well plate with adhesive cover (the worse version) and poke a hole through cover with a needle.
• Put on a shaker at 37°C at 300 RPM for 22 hours
• [in sterile hood] Prepare the 96-well plate with 80 µL with 50% glycerol
• [in sterile hood] Pipette 80 µL of bacteria and mix with a pipette with the glycerol. Cover the 96-well plate with (better) adhesive cover along with plastic cover. Place glycerol stock in -70°C freezer.
• [on ice] Centrifuge at 4000 RPM for 10 minutes (put RNAse on ice to thaw)
• [on ice] Discard the supernatant and turn plates upside down on paper towel
• [on ice] Add 240 µL of solution 1; seal plates with [worse] cover and resuspend with vortex.
• [on ice] Centrifuge at 4000 RPM for 10 minutes
• [on ice] Prepare solution 2
• [Preparing solution 2: not on ice] Add 5.58 µL of water
• [Preparing solution 2: not on ice] Add 120 µL of 10M NaOH and mix thoroughly
• [Preparing solution 2: not on ice] Finally, add 300 µL of 20% SDS and mix (make sure there isn’t any precipitate)
• [on ice] Discard the supernatant and turn plates upside down on paper towel
• [on ice] Add 80 µL of solution 1 – seal plate and resuspend cells with vortex
• [on ice] Take the U-bottom 96-well plate, and add 5 µL of RNAse (10 mg/mL) per well
• Transfer 60 µL of cells to the U-bottomed plate
• Add 60 µL of solution 2; seal with [better] cover and invert plate 10 times
• Leave on the bench for 10 minutes. Spin for a few seconds. (Turn on and preheat heater to 80°C)
• Add 60 µL of solution 3. Seal with [better] cover and invert 10 times
• Leave on the bench for 10 minutes. Spin for a few seconds.
• Take off cover and place plate in 80°C heater.
• [no adhesive cover] Put on ice for 10 minutes.
• [no adhesive cover] Centrifuge at 4000 RPM for 10 minutes
• [no adhesive cover] Attach V-bottom 96-well plate to plate with filter and secure using masking tape
• Transfer all the sample without the pellet and centrifuge at 4000 RPM for 15 minutes.
• Discard the filter plate and add 110 µL of cold 100% isopropanol
• Cover the plate with [better] cover and invert 10 to 20 times
• Spin, change the seal to another cover and centrifuge for 45 minutes (turn on speed vacuum)
• Discard the supernatant and add 200 µL of cold 70% ethanol
• Centrifuge for 5 minutes at 4000 RPM. Discard supernatant
• Dry the plate for 15 minutes using the speed vacuum
• Resuspend with 40 µL of water

To validate plasmid extraction, do a digest and a run a gel or cover and put in -20°C freezer.

---

Restriction Digest

We use BamHI, EcoRI, NotI, PstI, SpeI for the general reaction:

• NEB Enzyme X: 1 uL (for 10.000 enzimatic unit)
• NEB Enzyme Y (if is a double digestion): 1 ul
• corresponding NEB Buffer: 2 uL      
• If necessary BSA: 0.2 uL
• Water to 20 uL

---

PCR Reaction

PCR was carried out on ice with Taq pol Enzyme following the protocol:

Component	Volume	Final Concentration
10X PCR buffer minus Mg	10μL	1X
10 mM dNTPmixture	2μL	0.2 mM each
50 mM MgCl2	3μL	1.5 nM
Primer mix (10 μM each)	5μL	0.5 μM each
Template DNA	1-20μL	n/a
Taq DNA Polymerase (5 U/μl)	0.2-0.5μL	1.0–2.5 units
Autoclaved distilled water	to 100μL	n/a

Temperature cycles:

Temperature	Time
98 ° C	0:10
98 ° C	0:30
69 ° C	1:00
72 ° C	1:00 (and repeat 35x)
72 ° C	2:00
10 ° C	hold

---

Ligation

• pPIC9K: 10 ng/µL; 5 µL for 50 ng
• RFP: 17.2 ng/1.72 nl
• 16 ° C overnight

Ligation reaction	µL
2X buffer	10
pPIC9K	5
RFP	1.72
T4 ligase	1(and repeat 35x)
water	2.28

---

Competent Cells

Day 1:

• Streak out the E.coli onto an LB agar plate with streptomycin 12.5 mg / mL
• Incubate overnight at 37 ° C
• Autoclave 3-4 liters of milli-Q water

Day 2:

• Inoculate 5 mL of LB medium containing streptomycin 12.5 ug / mL (in a 50 mL Falcon: LB 5 mL + 1.25uL streptomycin 50 mg / mL) with a single colony
• Incubate 18 hours at 37 ° C at 200 rpm
• Put SOB media at at 37 ° C (to pre-warm)

Day 3:

• Take 4mL of the innoculant and add to 400 ml of SOB (no streptomycin) at 37 °C  .Incubate for 2 hours at 37 ° C at  200rpm
• Measure the OD600. The optimal OD is ~ 0.65. If you are below this value let the cells grow longer
• Upon reaching the desired OD, split the 400 mL into 8 x 50 mL tubes
• Incubate on ice for 20 minutes
• Centrifuge for 5 minutes at 7000g at 4° C. Discard the supernatant.
• Resuspend each pellet in 50 mL of cold autoclaved milli-Q H2O. In this step the total volume is 400 mL.
• Centrifuge for 5 minutes at 7000g at 4° C. Discard the supernatant.
• Resuspend each pellet in 25 mL of cold autoclaved milli-Q H2O.In this step the total volume is 200 mL.
• Put 2 tubes together into 1 tube, so you have a total of 4 tubes!
• Centrifuge for 5 minutes at 7000g at 4° C. Discard the supernatant.
• Resuspend the pellets in an 8 ml of autoclaved cold 10% glycerol. Meanwhiletheotherthree pellets are onthe ice!
• Resuspend the pellets remaining in the same solution, one at a time, gathering all the cells at the end! At the point you have 1 x 8mL tube.
• Centrifuge for 5 minutes at 7000g at 4° C (you will need a balance). Discard the supernatant.
• Resuspend in 1.2 to 1.6 mL (depending on size of pellet) of autoclavedta cold 10% glycerol.
• Make aliquots of 50 µL and immediately frozen in liquid N₂.
• Store in freezer at -80 ° C (yields about 48 eppendorfs).

Efficiency test:

• Dilute pGEM vector from 20ng/µL to 0.1 ng/µL; 0.2 ng/µL and 0.4 ng/µL
• 0.4ng/ µL = 1 µL of 20ng/ µL + 49 µL dH20;
• 0.2ng/ µL = 2 µL of 1ng/ µL + 2 µL dH20;
• 0.1ng/ µL = 2 µL of 1ng/ µL + 2 µL dH20
• Prepare 3 large LB+Ampicillin plates for 3 transformations (50mL per plate = 150mL total. Add 150 µL of ampicillin at a stock concentration of 100mg/mL)
• [each transformation] 1uL of the diluted pGEM + 5uLdH20
• [each transformation] Add all 6µL to an aliquot of competent cells and shock
• [each transformation] Add 950µL of SOC media. Incubate1 hour at 37° C.
• [each transformation] Dilute before plating out
• [each transformation] 10µL of culture (competent cells) + 990 µL of SOC
• [each transformation] Plate 100 µL onto large LB+Amp plate and incubate overnight at 37° C
• Next day: calculate efficiency
• Count number of colonies on plate
• Transformation efficiency (transformants/µg) is calculated as follows: # colonies on plate/ng of DNA plated X 1000 ng/µg 

---

Transformation of Competent Cells

Preparation:

• Place the cuvettes on ice (1 for processing)
• Prepare tubes (15mL Falcon tube or glass) containing 750μL of SOC medium (1 per transformation)
• Keep the SOC aliquot at room temperature (200μL by transformation)
• Thaw eletrocompetent bacteria aliquots on ice (approx. 2 min.)
• Connect the electroporation equipment(set pulse to 200 - machine above - and voltage to 2.5 kV)
• Aliquot DNA to be transformed (diluted with water or resuspended precipitate without salt and water if necessary)

Transformation:

• Mix bacterial DNA in eppendorf (approx. 50 to 100 ng for each 50mL of bacteria) and leave on ice for 1 min.
• Transfer the volume from the cuvettes, dry the metal sides and electroporate (press the two red buttons simultaneously and release when you hear the "beep"). NOTE: Do not hold the metal cuvettes by side and not eletroporate with moist. Dry before!
• IMMEDIATELY (In max. 30s) add 200μL of SOC to the cuvette and transfer the culture to the tube containing 750μL of SOC
• Incubate in shaker at 37°C for 40 minutes
• Centrifuge for 2min. at 4000rpm, discard until roughly half 150uL.
• Resuspend the samples.
• Plate on plates containing LB + appropriate antibiotic
• Incubate at 37°C for 18 hours.

---

Glycerol Stock for long-term storage of bacteria

• Prepare the 96-well plate with 80 ul with 50% glycerol
• Pipette 80 ul of bacteria and mix with a pipette with the glycerol. Cover the 96-well plate with adhesive cover along with plastic cover. Place glycerol stock in -70 ° C freezer.

---

Cell Cultures

---

Cell line and cultivation

• Escherichia coli: strain DH10B, cultivated at 27 ° C and 250 rpm, in a medium with the antibiotic correspondent to its plasmid.
• Pichia pastoris: strain GS115(his4), cultivated at 30 ° C

---

Transfection of Pichia pastoris

Transformation was performed by electroporation following the protocol:

• Keep electroporation cuvettes at -20 ° C;
• Gently mix 80μL of competent cells with 5-20μg of digested DNA (10μL).
• Transfers all mix to 0,2cm cuvettes (for electroporation). (Be careful: do not allow bubbles formation, electric current will pass from outside instead of inside in this case)
• Incubate cuvettes with yeast on ice for 5 to 10 minutes;
• Pulse cells following instructions of electroporation machine: Voltage 1500V,2. - Capacitance 25uF, Resistance 200.
• Add 1 mL of sorbitol 1M in each cuvette and transfer to a sterile eppendorf, in case of selection by gentamicin add 1mL of YPD media with 1M of sorbitol and incubate for 1h at 30°C;
• Plate 100 μL in a MD plate, 100 μL in a YPD plate with gentamicin and 100 μL in a YPD plate. Plate the remnant volume (800μL) in other MD plate (optional).
• Keep plates drying in sterile hood.
• Keep plates at incubator for 2 to 4 days at 30 ° C (verify growth daily).

---

Genomic DNA extraction

Genomic DNA was extracted from Pichia pastoris by the following protocol: Single-tube LiOAc-SDS lysis

• Collect 100 μL liquid YPD culture (OD600 = 0.4) or pick P. pastoris (or collect overnight culture in 5 mL)
• Suspend in 100 μL 200 mM lithium acetate (LiOAc) 1% SDS solution
• Vortex and incubate for 10 min at 70°C (or 20 min at room temperature)
• Add 300 μL of 96% ethanol (cold) for DNA precipitation
• Briefly vortex and collect DNA by centrifugation (15,000× g) for 3 min
• Remove supernatant and wash the pellet with 500 μL 70% ethanol (cold) (pipette up and down)
• Briefly vortex and collect DNA by centrifugation (15,000×g) for 3 min. Discard supernatant. Briefly dry pellet at room temperature​.
• Suspend in 100 μL water or TE to dissolve DNA
• Remove debris by centrifugation (15,000× g for 1 min)
• Use 1μL supernatant for PCR ​​

  ​OBS: To test gDNA quality, dilute 80-100 μL in 10-fold of water. Scan in spectrophotometer (200-360 nm) and analyse data.