Team:HKUST/Protocols

1. Agarose gel preparation and gel electrophoresis

  Purpose: To check the result
  Materials: Agarose, 0.5X TBE, SYBR safe DNA gel stain, DNA loading dye, DNA ladder/marker 
  Choice of ladders: VC100bp Plus DNA ladder (highest band 3000bp); 
                     Lamda DNA BstEII marker (highest band 8400bp, 1% argarose)
  Procedure: 
  a.To make a 0.8% agarose gel, use 0.16 g agarose per 20 mL 0.5x TBE. 
  b.Cover the flask with plastic wrap to prevent boiling over, then microwave the solution for 1-2 minutes to   dissolve the agarose. 
  c.Let agarose solution cool for 5 minutes, then add SYBR safe DNA gel stain to a final concentration of 0.5 μg/mL. 
  d.Pour the agarose solution into a casting tray with well comb in place. Allow 20-30 minutes to completely solidify.
  e.Place the agarose gel into the gel box and fill the box with 0.5X TBE until the gel is immersed.
  f.Load an appropriate molecular weight ladder into the first lane of the gel. If needed, an additional ladder with different molecular weight could be loaded into the last lane.
  g.Add 1 μL loading dye per 5 μL of sample.
  h.Load the samples from left to right.
  i.Cover the gel box and plug in the electrodes in a way that the samples will run towards the positive, red electrode.
  j.Run the gel at 100V until the dye line is approximately 50-75% of the way down the gel. Approximately 20-30 minutes.
  k.Carefully remove the gel from the gel box and check the result under UV exposure.
  Tips: 
  •Higher concentration of agarose solution makes better resolution for less molecular weight expected band.
  •Let bottom of the flask be immersed in a cup of cold water for faster cooling.
  •In order for clearer band to cut specific band on the gel, immerse the gel in Ethidium bromide for 10-15 minutes after step k.
  Safety tips: 
  •Be sure to wear a glove before treating the hot flask.
  •Ethidium bromide is a strong mutagen. Wear a lab coat, eye protection, and gloves when working with this chemical.

2. PCR

  Purpose: To amplify a specific piece of DNA out from the whole. 
  Materials: ddH2O, 10X ThermoPol Buffer, 1mM dNTP, Forward primer, Reverse Primer, Taq or Vent DNA polymerase, DNA template
             Choice of DNA polymerase: Taq gives higher rate of mutation compared with Vent.
                                       Taq gives sticky ends after PCR, while Vent gives blunt ends.
  Procedure: 
  a.Add 10 μL water to make it a 20 μL reaction.
  b.Add 2 μL buffer, 4 μL dNTP, 1 μL forward primer (5 μM), 1 μL reverse primer (5 μM), 1 μL DNA template, 1 μL DNA polymerase
  c.Vortex and then spin down.
  d.Put it into the PCR machine and set the program.
    Program
    (1) Initial denaturation   95 °C   4 mins
    (2) Run 25-30 cycles of:
        Denaturation           95 °C  30 secs
        Annealing                     30 secs 
                               Temperature is depended on melting temperature of primer. 
                               Lower Tm-5°C, for Taq; Lower Tm +3°C for Vent
        Extension              72 °C   30 secs per 500bp PCR product
    (3) Final extension        72 °C   3~5 mins
  Tips: 
  •The DNA polymerase is easy to be denatured, so do not leave it at room temperature. It is highly recommended to use an ice holder or do the adding in the fridge.

3. PCR product clean-up

  Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.
  Materials: FAPC Buffer，Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR Clean-Up Kit)
  Procedure:
  a.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.
  b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.
  c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.
  d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.
  e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.
  f.Place FAPC Column into an Elution Tube.
  g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.
  h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.
  Tips:
  •In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.

4. Enzyme digestion

  Purpose: To cut the sequence with specific restriction enzyme
  Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)
  Procedure: 
  a.Add sufficient water to make it a 20 μL reaction.
  b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.
  c.Vortex and then spin down.
  d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)
  e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.
  f.Check the result by gel electrophoresis.
  Tips: 
  •Restriction enzymes are easy to be denatured, so do not leave it at room temperature.
  •If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.

5. Plamid DNA extraction

  Purpose: To extract plasmid DNA from E.coli.
  Materials: MX1 Buffer, MX2 Buffer, MX3 Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in Mini Plus Plasmid DNA Extraction System)
  Procedure:
  a.Grow 1 to 5 ml plasmid-containing bacterial cells in LB medium with appropriate anlibiotic overnight (12-16 hours) with vigorous agitation.
  b. Pellet the cells by centrifuging for 1-2 minutes. Decant the supernatant and remove all medium residues by pipette.
  c.Add 200 μL of MX1 Buffer 10 the pellet, and resuspend the cells completely by vortexing or pipetting.
  d.Add 250 μL of MX2 Buffer and gently mix well (invert the tube 6-10 times) to lyse the cells until the lysate becomes d ear. Incubate at room temperature for 2-5 minutes
  e.Add 350 μL of MX3 Buffer to neutralize the lysate, then immediately and gently mix the solution well. A white precipitate should be formed.
  f.Centrifuge at 10,000 x g (13,000rpm) for 5-10 minutes, meanwhile place a Mini Plus™ Column onto a Collection Tube. Transfer the supernatant carefully into the column.
  g.Centrifuge at 7,000x g (9,000rpm) for 30-60 seconds. Discard the flow-through.
  h.Wash the column once with 0.5 ml of WN Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.
  i.Wash the column once with 0.7 ml of WS Buffer by centrifuging at 7,000 x g (9,000rpm) for 30-60 seconds. Discard the flow-through.
  j.Centrifuge the column at 10,000 x g (13,000rpm) for another 3 minutes to remove residual ethanol.
  k.Place the column into a new 1.5-ml centrifuge tube. Add 50 μL of Elution Buffer onto the center of the membrane.
  l.Stand the column for 2-3 minutes, and centrifuge at 10,000 x g (13,000rpm) for 2-3 minutes to elute DNA. Store plasmid DNA at 4 °C or -20 °C.
  Tips: 
  •In step c, no clump should be visible after resuspension. Clumped cells lead to bad plasmid yield and quality.
  •Do not vortex in step d, otherwise genomic DNA will be sheared and contaminate the sample, which can be observed as serious foaming.
  •In step d, the lysate should become clear and viscous. Insufficient cell-lysis leads to low plasmid yield and quality.
  •MX1 Buffer must be stored at 4 °C.

6. Cutting bands on the gel

  Purpose: To extract specific DNA fragment.
  Procedure: 
  a.Visualize the bands under UV light. Use long-wavelength UV to minimize damage to the DNA.
  b.Cut the band with a clean razor blade.
  c.Turn the gel slice on its side to trim off extra agarose. Place the gel in a microcentrifuge tube.
  Tips:
  •EB staining is needed for higher resolution under UV light.
  Safety Tips: 
  •Permission is needed from technician before doing this! Follow the safety instruction in the room.
  •When using UV light, protect your skin by wearing safety goggles or a face shield, gloves, and a lab coat.

7. Gel extraction

  Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.
  Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit)
  Procedure:
  a.Add 0.5ml GEX buffer into the tube with gel fragment in it. 
  b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.
  c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.
  d.Repeat step c for the excessive gel mixture.
  e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.
  f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).
  g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol
  h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).
  i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.
  Tips:
  •The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.

8. Ethanol precipitation

  Purpose: To concentrate DNA product.
  Materials: 100% ethanol, 75% ethanol, 3M NaAc, ddH2O, DNA product
  Procedure:
  a.Add 2 volume of 100% ethanol.
  b.Add 0.1 volume of 3M NaAc.
  c.Put the tube in fridge at -20 °C for 10 minutes.
  d.Put it out and centrifuge it for 10-15 minutes (13,000 rpm). Remove the supernatant.
  e.Wash with 100μL 75% ethanol.
  f.Centrifuge for 5 minutes (13,000 rpm). Remove all the supernatant.
  g.Add 10μL ddH2O to resuspend DNA.
  Tips:
  •If the volume of DNA product is too low, make it to higher volume with ddH2O. The recommended lowest volume is 50μL. Combine several tubes of DNA product is also suggested for higher DNA product concentration.
  •In step f, make sure to remove all the supernatant without touching the pellet. If it is too hard to do so, open the tube and leave it at room temperature for a while to make the ethanol evaporate.

9. Ligation

  Purpose: To ligate desired insert DNA fragment into vector with specific restriction site.
  Materials: digested insert DNA and plasmid DNA with the same restriction site, T4 DNA ligase, 10X T4 Ligation Buffer, CIP (Calf Intestinal Alkaline Phosphatase), ddH2O
  Procedure: 
  a.Add sufficient water to bring to a total volume of 10 μL or 20 μL, depending on the volume of the two DNA pieces.
  b.Add 50ng of vector DNA.
  c.Add enough amount of insert DNA, which can be calculated by ligation calculator online.
  d.Add 2μL ligation buffer and 0.5μL ligase.
  e.Incubate at 16 °C overnight or at room temperature for 8 hours.
  Tips:
  •Ligase blank control group is recommended.
  •The plasmid DNA and insert DNA for ligation must be added with CIP when enzyme digestion.

10. Transformation to yeast

  Purpose: To transform desired constructed plamid DNA to yeast to test its effect.
  Materials: LiOAc solution, PEG4000 solution, DMSO (dimethyl sulfoxide), yeast strain, desired plasmid, YEPD, SD media with specific selection marker absent (-Leu, -His, or -Ura), ddH2O
  Procedure:
  a.A stationary culture of yeast grown in YEPD is used to inoculate 10mL of YEPD in a 100mL Pyrex flask.
  b.Cells are grown at 30 °C with shaking (200 rpm) until a density of 1-4x107 is reached (OD660=0.4).
  c.Yeast is transformed into 4 sterile 1.5mL tubes and are centrifuged for 2mins (4,000 rpm).
  d.Damp off media and wash the pellet with 100μL ddH2O (to culture 1:1). Resuspend the cells by gently shake it or flip it.
  e.Centrifuge it again for 2mins (4,000 rpm). Remove the supernatant.
  f.Wash the pellet with 1mL LiOAc solution and pour it into a single tube. Resuspend the cell by gently shake it or flip it. 
  g.Repeat step e and f once.
  h.Add 100μL of yeast suspension to a 1.5mL microcentrifuge tube, and add 10μL of DNA to be transformed. Mix them gently and stand the tube at room temperature for 5mins.
  i.Add 280μL PEG4000 solution. Mix it gently by inverting 4-6 times, then the tube is stored at 30 °C for 45mins without shaking.
  j.Add 43μL DMSO to give an approximate 10% (volume percentage) DMSO solution. Mix the solution gently by inverting.
  k.Heat shock at 42°C for 5mins.
  l.Centrifuge it for time long enough to get pellet (usually 5 sec) at 12,000rpm.
  m.Remove the supernatant and wash it with ddH2O.
  n.Centrifuge it again for 50sec (13,000 rpm). Remove the supernatant.
  o.Resuspend the cell with 1mL ddH2O.
  p.Plate the solution on SD media with specific selection marker absent.
  Tips:
  •Ignite the fire for sterile environment when transforming the yeast cell, since there is no any antibiotic in the culture.
  Safety Tips:
  •Be careful with the fire, and extinguish it after use.

11.Transformation to E.coli

  Purpose: To transform desired plasmid DNA in to E.coli in order to amplify it.
  Materials: E.coli competent cell, desired plasmid DNA, LB media, /agar plate with desired selection antibiotic (ampicillin)
  Procedure:
  a.Perform ligation or any other manipulation of DNA to yield circularized plasmids.
  b.Remove bacterial aliquots (usually the DH5 strain of E.coli) from -70C freezer and thaw on ice for 10-15 min; briefly flick the tube to resuspend the bacterial cells, but minimize any time the cells are not on ice.
  c.Add desired amount of plasmid DNA (usually 1-5μL of a standard ligation or 1.0ng of purified plasmid) to the bacteria at the bottom of the tube, mix it with pipette and incubate 5min on ice.
  d.Remove the tube from ice and immediately begin heat shock in a water bath at 42C for 45 sec.
  e.After heat shock, immediately add 500μL of room temperature LB media to the bacterial cells, then incubate in a 37 °C shaker incubator for 45mins.
  f.Plate 50μL of the bacterial culture to a LB/agar plate with desired selection antibiotic (ampicillin) using a sterile bacterial streaking rod, and allow the plate to absorb the extra liquid for 15 min on the benchtop at room temperature.
  g.Invert the plates and place in the 37C bacterial incubator overnight.
  Tips:
  •The competent cell is quite fragile, so make sure that it does not leave the ice before heat shocking.
  •LB is easily contaminated, make sure to sterile it with fire each time when opening or closing it. Add it quickly to minimize the time of leave it open.
  •Ignite the fire for sterile environment when adding LB.
  Safety Tips:
  •Be careful with the fire, and extinguish it after use.

12.Cell lysis

  Purpose:  to lyse E.coli cell to get linearized ligated plasmid.
  Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O
  Procedure: 
  a.Add 40μL ddH2O in a 1.5mL microcentrigfuge tube.
  b.Use a toothpick to pick a colony and dissolve it in the water.
  c.Add 20μL phenol and 20μL chloroform.
  d.Scrape the tube to mix the solution. A well mixed white solution can be seen after this step.
  e.Centrifuge it for 10mins (13,000 rpm).
  f.Place the supernatant in another tube.
  g.Load 10μL to test the result by gel electrophoresis.
  Tips:
  •Ignite the fire for sterile environment when transforming the colony.
  Safety Tips:
  •Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.

B •Be careful with the fire, and extinguish it after use. 13.Yeast genomic DNA extraction

  Purpose: To extract yeast genomic DNA from yeast strain (strain YPH501, in our case).
  Materials: yeast strain, lysis buffer, phenol, chloroform, 3M NaAc, 100% ethanol, 70% ethanol, ddH2O, glass beads.
  Procedure:
  a.Add 200μL lysis buffer.
  b.Pick a whole patch of colony and add it in the tube.
  c.Add 100μL phenol, and mix it well.
  d.Add 100μL chloroform, and a few glass beads.
  e.Vortex for 4-5mins and centrifuge for 5mins (13,000 rpm). 
  f.Put the supernatant (~160μL) to another tube. 
  g.Add 200μL phenol/chloroform (1:1 in volume)
  h.Vortex for 4-5mins and centrifuge for 5mins (13,000 rpm). 
  i.Put the supernatant (~120μL) to another tube. 
  j.Add 0.1 volume NaAc(pH5.2), approximately 12μL.
  k.Add 2 volume of 100% ethanol, approximately 240μL.
  l.Store it at -20C for 20mins.
  m.Centrifuge for 10mins (13,000 rpm) and remove the supernatant.
  n.Add 2 volume of 70% ethanol.
  o.Centrifuge for 5mins (13,000 rpm) and remove the supernatant. 
  p.Stand the tube without closing it for a while to let the ethanol evaporate.
  q.Add 20μL ddH2O to resuspend it.
  r.Store it at -20 °C.
  Safety Tips:
  •Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.