Team:USP-Brazil/Protocols

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Problem

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

Reagents1 reaction
ECO RI*1μL
Enzyme Buffer2μL
DNA2μL
H2O15μL
Total20μL
* place the enzyme last

Solutions P1, P2 and P3

P1)

  • 23 ml glucose 20%
  • GTE 500 ml
  • 10 ml EDTA 0.5 M pH 8,0
  • 12.5 ml of Tris HCl 1M pH 7,0
  • Water to 500 ml
  • Autoclave before using

P2)

  • H2O 5580 ul
  • NaOH 120 ul
  • 300 ul SDS 20%

P3)

  • KaOH 3M
  • 60 ml KOAC 5M
  • 11.5 ml Glacial Acetic Acid
  • Water to 100 ml
  • 11.5 ml Glacial Acetic Acid
  • 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:

ComponentVolumeFinal Concentration
10X PCR buffer minus Mg10μL1X
10 mM dNTPmixture2μL0.2 mM each
50 mM MgCl23μL1.5 nM
Primer mix (10 μM each)5μL0.5 μM each
Template DNA1-20μLn/a
Taq DNA Polymerase (5 U/μl)0.2-0.5μL1.0–2.5 units
Autoclaved distilled waterto 100μLn/a

Temperature cycles:

TemperatureTime
98 ° C0:10
98 ° C0:30
69 ° C1:00
72 ° C1:00 (and repeat 35x)
72 ° C2:00
10 ° Chold

Ligation

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

Ligation reactionµL
2X buffer10
pPIC9K5
RFP1.72
T4 ligase1(and repeat 35x)
water2.28

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.