Team:NEU-China/Protocol

Protocol - NEU-China

Protocol

List of some kits and reagents

List of experimental kits

List of biological reagents

kits

origin

anti-Ha, anti-Flag antibody

Sigma,Abcam
Plasmid Mini Kit I(200) Omega anti-rabbit IgG Proteintech
Plasmid Mini Kit(25) Omega anti-mouse IgG Proteintech
Cycle-Pure Kit(200) Omega GoTaq Colonrless Master Promega
Gel Extraction Kit I(200) Omega PrimerSTAR Max DNA Polymerase Takara
Endo-free Plasmid Mini Kit II(50) Omega PrimerSTAR HS DNS Polymerase Takara
GoScript Reverse Transcription System Promega DL2000 DNA Marker Takara
QIAquick PCR Purification Kit(250) QIAGEN 1Kb DNA Ladder(Dye Plus) Takara
QIAquick Gel Extraction Kit I(250) QIAGEN 6x DNA Loading Buffer Takara
In-Fusion HD Cloning Kit Takara 10x DNA Loading Buffer Takara
DNA Ligation Kit Ver.2.1 Takara Protease Inhibitor Cocktail CWBIO
Blunting Kination Ligation Kit Takara 2x Es Taq Master Mix CWBIO
BCA Protein Assay Kit CWBIO Protease Inhibitor 8340 Sigma
cECL Western Blot Kit CWBIO PMSF Sigma
eECL Western Blot Kit CWBIO Prestained Protein Ladder Thermo
Plant RNA kit OMEGA Xma I, Restriction enzyme NEB
Apa I, BamH I, EcoR I, , Kpn I, Not I, Sal I, Xba I, Spe I, Pst I, Xba I Takara

Some essential chemical reagents

SDS, acrylamide, TRIS Base, ammonium peroxydisulfate, HCl, NaCl, TEMED, methanol, Triton-X 100, EDTA,ammonium acetate, Glycine, chloroform, isopropanol, ethanol, HEPES, UltraPure Phenol, Calcium chloride, Ampicillin, YPD Broth Medium, YPD Agar Medium, LB Broth Agar Medium, 2,2,2-Tribromoethanol, 2-Methyl-2-butanol, etc.

  • Hepes lysis buffer：
    50mM HEPES, 140mM NaCl, 1%Triton-X 100, 10mM EDTA;
    The usage of protease inhibitor: Add 10ul protease inhibitor and 25ul PMSF(phosphatase inhibitor) into 1ml HEPES lysis buffer.
  • Yeast DNA lysis buffer:
    0.025g EDTA, 0.5844g NaCl, 2ml Triton-X 100, 10ml 10% SDS, dilute with deionized water to 100ml.
  • 10x Running buffer:
    30.3g Tris-Base, 144gGlycine, 10g SDS, dilute with ddH2O to 1000ml, mix thoroughly.
  • Blotting buffer:
    10x Blotting buffer:
    30.3g Tris-Base, 144gGlycine, dilute with ddH2O to 1000ml, mix thoroughly.
    1x Blotting buffer:
    100ml 10x Blotting buffer, 700ml ddH2O and 200ml methanol.
  • SC liquid culture medium:
    6.7g Yeast Nitrogen Base 6.7g, 2g SC, 20g glucose, dilute with ddH2O to 1000ml, sterilize within high heat and pressure.

Plates:

Amino Acid Mix (40X) Use 0.6 g/liter:

  • Tyrosine 2g
  • Arginine 4g
  • Serine 2g
  • Valine 2g
  • Threonine 4g
  • Isoleucine 2g
  • Phenylalanine 2g
  • Aspartic Acid 2g
  • Proline 2g

Glucose Complete Plates (1 liter):

  • 1.7 g Yeast Nitrogen base -amino acids and ammonium sulfate
  • 5.0 g Ammonium Sulfate
  • 1 liter H2O
  • pH to 7.0 using NaOH

Add:

  • 0.6 g Amino Acid mix (mix well before adding)
  • 20g Bacto-agar (Difco)
  • Autoclave 25 min.

Add:

  • 10ml (10/mg/ml) of any other required amino acids
  • 10ml 0.4% Adenine sulfate in 0.1M HCl if required
  • 10 ml 0.2% Uracil if required
  • 50 ml 40% glucose

YPD Plates (1 liter):

  • Yeast Extract 10 g
  • Bacto peptone 20g
  • Bacto-Agar 20g
  • H2O 1 liter
  • Autoclave 25 min

Add:

  • 50 ml 40% glucose
  • 5 ml 0.4% Adenine sulfate in 0.1 M HCl if required

Add 10 ml 0.2% Uracil (only if plates will be used for tetrad dissection)

Protocal:

Extract the plant RNA:

Method: Plant RNA kit,E.Z.N.A.® Plant RNA Kit Standard Protocol

  1. Collect tissue in a 1.5 mL microcentrifuge tube and freeze by dipping in liquid nitrogen. Grind the tissue using disposable pestles.
  2. Transfer up to 100 mg frozen ground plant tissue to a new 1.5 mL microcentrifuge tube
  3. Immediately add 500 μL RB Buffer. Vortex at maximum speed to mix thoroughly.
  4. Insert a Homogenizer Column into a 2 mL Collection Tube.
  5. Transfer the lysate to a Homogenizer Column.
  6. Centrifuge at 14,000 x g for 5 minutes at room temperature.
  7. Transfer cleared lysate to a new 1.5 mL microcentrifuge tube. Do not disturb or transfer any of the insoluble pellet. Measure the volume of the lysate.
  8. Add 1 volume 70% ethanol. Vortex at maximum speed for 20 seconds. A precipitate may form at this point; it will not interfere with DNA isolation. Passing the mixture through a needle using a syringe or by pipetting up and down 10-15 times may break up the precipitates.
  9. Insert a HiBind® RNA Mini Column into a 2 mL Collection Tube.
  10. Transfer 700μL sample, including any precipitates that may have formed, to the HiBind® RNA Mini Column.
  11. Centrifuge at 12,000 x g for 1 minute at room temperature.
  12. Discard filtrate and reuse the collection tube.
  13. Repeat Steps 10-12 until all of the sample has been transferred to the column.
  14. Add 500 μL RNA Wash Buffer I.
  15. Centrifuge at 10,000 x g for 30 seconds.
  16. Discard the filtrate and the collection tube.
  17. Transfer the HiBind® RNA Mini Column to a new 2 mL Collection Tube.
  18. Add 700 μL RNA Wash Buffer II.
  19. Centrifuge at 10,000 x g for 30 seconds.
  20. Discard the filtrate and reuse collection tube.
  21. Add 500 μL RNA Wash Buffer II.
  22. Centrifuge at 10,000 x g for 30 seconds.
  23. Discard the filtrate and reuse collection tube.
  24. Centrifuge the empty HiBind® RNA Mini Column at maximum speed for 2 minutes.
  25. Transfer the HiBind® RNA Mini Column to a clean 1.5 mL microcentrifuge tube.
  26. Add 50-100 μL DEPC Water.
  27. Centrifuge at maximum speed for 1 minute and store eluted RNA at -70°C.

Notice: steps 14-23 are expected to be done if you want to remove the genome gene.

RT-PCR

Method:GoScript Reverse Transcription System
Protocal:

  1. microcentrifuge the reagents of kit
  2. Prepare mix A as follows:
    • RNA: x ul ,make sure the RNA content is up to 1ug/ul.
    • oligdT: 1 ul
    • dNTP: 1 ul
    • DEPC water: (8-x) ul
    • Total: 10 ul
  3. Incubate mix A for 5 minutes at 70℃ in PCR machine. Then, set on the ice for 5min.
  4. Prepare mix B as follows:
    • MgCl2: 2ul
    • DEPC water: 1.6ul
    • GoScript: 4ul (5x Reaction Buffer)
    • NucMix: 1ul
    • RNase: 0.4ul
    • RTE: 1ul
    • Total: 10ul
  5. Add mix B to mix A. Set PCR procedure:

    6. 25℃ 5minutes → 42℃ 60minutes → 70℃ 15minutes → 4℃ ∞
    Insert PCR tube with mixed system into PCR machine, store at -20℃。

Standard PCR Protocal:

  1. Add DNA template up to 50ng into PCR tube,(x ul).
  2. Add reagents as follow:
    • dNTP: 4ul
    • 5x PSBuffer: 10ul
    • Primer: 1ul (after diluted)
    • Enzyme: 0.5ul
    • ddH2O: (33.5-x)ul
    Total: 50ul
    Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.
  3. PCR procedure:
    98℃(2min)→ 98℃(10s)→55℃(5s)→72℃(1Kb/min)→72℃(5min)→4℃(∞)
  4. The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.

PCR purification：

Method:Cycle-Pure Kit(200)

  1. Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.
  2. Determine the volume of the PCR reaction. Transfer the sample into a clean 1.5ml microcentrifuge tube and add 4-5 volumes of Buffer CP. For PCR products smaller than 200bp, add 6 volumes of Buffer CP.
  3. Vortex thoroughly to mix. Briefly spin the tube to collect any drops from the inside of the lid.
  4. Place a HiBind DNA Mini Column into a provided 2 ml collection tube.
  5. Add the mixed sample from step 3 to the HiBind DNA Mini Column and centrifuge at 13,000 x g for 1 minute at room temperature. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
  6. Add 700µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
  7. Add 500µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
  8. Centrifuge the empty HiBind DNA Mini column for 2 min at maximal speed ( ≥13,000 x g ) to dry the column matrix.
  9. Place the HiBind DNA Mini column into a clean 1.5ml microcentrifuge tube. Depending on the desire concentration of the final product, add 30-50 µl of Elution Buffer (10mM Tris, pH8.5) or water directly onto the center of column matrix.

OVERLAP PCR:

  1. order two primers which are complements of one another.
  2. These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
  3. The "end primers" will not have any complements and will likely only have restriction sites.
  4. PCR amplify the necessary fragments separately. Use a proofreading polymerase enzyme. Use an annealing temp of 60°C.
  5. Clean up the product using a DNA column.
  6. "Overlap PCR" Use cleaned up fragments as template in a PCR reaction:
  7. About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments. Do not use Phusion polymerase. Try Pfu Turbo. Do not add any primers; the templates will prime each-other. 3.Run 15 PCR cycles without primers.Use an annealing temp of 60°C.
  8. Add end primers to the Overlap PCR reaction: Continue cycling for another 15-20 rounds. Use an annealing temp of 72°C
  9. Gel extract the correct size fragment.

Gel Extraction

Method:QIAquick Gel Extraction Kit I(250)

  1. Excise the gel slice containing the DNA band with a clean, sharp scalpel. Minimize the size of the gel slice by removing excess polyacrylamide.
  2. Weigh the gel slice. Add 1–2 volumes of diffusion buffer to 1 volume of gel(i.e., 100–200 µl for each 100 mg of gel).
  3. Incubate at 50°C for 30 min.
  4. Centrifuge the sample for 1 min.
  5. Carefully remove the supernatant using a pipet or a drawn-out Pasteur pipet. Pass thesupernatant through a disposable plastic column or a syringe containing either aWhatman GF/C filter or packed, siliconized glass wool to remove any residualpolyacrylamide.
  6. Determine the volume of the recovered supernatant.
  7. Add 3 volumes of Buffer QG to 1 volume of supernatant and mix. Check that the color of the mixture is yellow.
  8. Place a QIAquick Spin Column in a provided 2 ml collection tube.
  9. To bind DNA, apply the sample to the QIAquick Spin Column and centrifuge for30–60 s.
  10. Discard flow-through and place QIAquick Spin Column back into the same collection tube.
  11. To wash, add 0.75 ml Buffer PE to column and centrifuge for 30–60 s.
  12. Discard flow-through and place QIAquick Spin Column back in the same tube.
    Centrifuge column for an additional 1 min at maximum speed. 
  13. Place QIAquick Spin Column into a clean 1.5 ml microcentrifuge tube.
  14. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick Spin Column and centrifuge for 1 min. 

Plasmid Extraction:

Method:Plasmid Mini Kit I(200)

  1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture.
  2. Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate and discard the culture media.
  3. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
  4. Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.
  5. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
  6. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
  7. Transfer the cleared supernatant from Step 5 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.
  8. Centrifuge at 10000g speed for 1 minute.
  9. Discard the filtrate and reuse the collection tube.
  10. Add 500 µL HB Buffer.
  11. Centrifuge at 10000g speed for 1 min.
  12. Discard the filtrate and reuse collection tube.
  13. Add 700 µL DNA Wash Buffer.
  14. Centrifuge at 10000g for 1 minute.
  15. Discard the filtrate and reuse the collection tube.
  16. Centrifuge the empty HiBind® DNA Mini Column for2 min at 13000g to dry the column matrix.
  17. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
  18. Add 30-50 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
  19. Let sit at room temperature for 5 minute.
  20. Centrifuge at 13000g for 1 minute.
  21. Store DNA at -20°

Yeast Transformation:

PREPARATION OF YEAST COMPETENT CELLS:

  1. dilute an overnight yeast culture 1:20 in 50ml YPAD broth to an OD600 of 0.25(for 10 transformation).
  2. Incubate the culture at 30℃ for approximately 4-5 hours until it reaches an OD of 1.0.
  3. Centrifuge the cells at 1000xg for 5 min. Discard the supernatant.
  4. Resuspend the cells in 10ml of LTE buffer.
  5. Centrifuge the suspension at 1000xg for 5 min. Discard the supernatant.
  6. Resuspend the cell in 0.5ml of LTE buffer.
  7. Aliquot 50ul of the cell suspension int microcentrifuge tubes.

TRANSFORMATION OF WHOLE YEAST CELLS
Prepare the transformation mix and amino acid dropout plates prior to beginning transformation of whole yeast cells.

  1. Add 1-3ug of the chosen plasmid or its derivatives.
  2. Add 300ul of the transformation mix to each tube. Invert to mix thoroughly.
  3. Incubate the tubes at 30℃ for 30 min.
  4. Heat the tube at 42℃for 15 min.
  5. Plate 100ul and 250ul respectively of the transformation reaction onto plates.

Note:
LTE Buffer:

  • 0.1M LiOAc
  • 10mM Tris-HCl(PH7.5)
  • 1mM EDTA

Transformation Mix:

  • 40% polyethylene glycol 3350
  • 0.1mM LiOAc
  • 10mM Tris-HCl(PH7.5)
  • 1mM EDTA

E.coli Competent cell:

  1. Innoculate a single colony of E. coli into 5 ml LB and grow O/N at 37
  2. Innoculate 1 ml into 100ml and grow to an O.D. 600 of 0.4
  3. Aliquot the culture into 2x 50ml pre-chilled Sorvall tubes and leave on ice for 5 -10 mins
  4. Centrifuge cells for 7 mins at 3000rpm, 4℃ without brakes
  5. Pour off supernatant and resuspend each pellet in 10 ml of ice-cold CaCl2 soln
  6. Centrifuge cells for 5 mins at 2500rpm, 4 degree. Discard supernatant and
    7. resuspend each pellet in 10 ml of cold CaCl2 solution.
  7. Keep resuspended cells on ice for 30min.
  8. Centrifuge cells for 5 mins at 2500rpm, 4℃. Discard supernatant and resuspend each pellet in 2ml of ice-cold CaCl2.
  9. Dispense cells into pre-chilled sterile eppendorfs.
  10. Freeze immediately @ -70 degresss

Notice: CaCl2 Soln: 60mM CaCl2, 15% Glycerol

Yeast Protein Extraction

Method:TCA

  1. Harvest cells, spin at 3000 rpm, 4 degree for 3 min
  2. Wash with 5 ml of water
  3. Add 5ml of 2% Kac, invert to mix
  4. Add 1 ml of 100% TCA, keep on ice
  5. Spin again, aspirate
  6. Resuspend in 1ml 10% TCA and trasnfer to 1.5 ml epp tube
  7. Sipn and aspirate
  8. Snap freeze in liquid nitrogen
  9. Resuspend in 200 ul of 10% TCA and transfer to a screw cap tube which contains 200ul of glass beads
  10. Vibrax in bead beater at 4 degree for 1 min
  11. Pipette extract from beads and wash beads with 200 ul 10% TCA and pool supernant with extract
  12. Spin at 3000 rpm at 4 degree for 10min to get large white pellet, and aspirate
  13. Add 100 ul of 1Xlaemmli buffer, mix to get yellow sample
  14. Add 25ul of 1 M tris pH8 and mix to get dark blue sample
  15. Boil at 95 degree for 10 min
  16. Spin at 3000 room temperature for 10 min
  17. Transfer supernatant to fresh tube
  18. Prepare BSA (NEB 10mg/ml). Make 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, 0.625mg/ml solution.
  19. Dilute Biorad protein assay 1:5with water.
  20. 2ul of sample, blank, and BSA + 18ul of water, and add 10ul of this mixture into 990ul diluted Biorad protein assay solution. Read at OD595nm
  21. Normal extraction efficiency 1 mg/ml to 10mg/ml

Coomassie Blue Staining

Materials and Reagents:

  1. Coomassie Brilliant Blue R250 (EM Science)

Equipment:

  1. Shaker

Procedure:

  1. Incubate the gel in staining solution with shaking for 30 min or longer (can leave it overnight).
  2. Remove the dye solution (it can be reused for many times) and rinse the gel with water 1-2 times to remove the dye.
  3. Add destaining solution to the gel and incubate for 30-60 min.
  4. Transfer the gel to water (can keep it in water for several days)

Recipes:

  1. 100 ml Staining solution
    • Coomassie Brilliant Blue R250 0.25 g
    • Glacial acetic acid 10 ml
    • MetOH:H2O (1:1 v/v) 90 ml
  2. Destaining solution

    3. Destaining solution is the same as staining solution, but not containing the Coomassie R250 dye Powder.

Silver Staining

  1. Fix gel in 10% MeOH / 10% Acetic Acid for 30 min or overnight
  2. Wash 4X in H2O for at least 5 min
  3. Incubate in sodium thiosulfate (1-2 pellets [400mg] per 500 ml) for 90 sec (Save 20 ml for later)
  4. Wash 3X quickly with H2O
  5. Add Silver nitrate solution (0.9 g silver nitrate in 500 mL H2O) for 10 min. Gel will turn slightly yellow
  6. Wash the gel 3X quickly in H2O
  7. Add developer solution (10g potassium carbonate, 20 ml sodium thiosulfate, 250 μl 40% Formaldehyde in 500 ml)
  8. Stop the reaction by adding destain (10% MeOH and 5% Acetic Acid)
  9. Wash gel in water

Western blot protocal:

Put blot into antibody:

  1. Remove blot from fridge.
  2. Prepare primary antibody:
    • 10mls TBS
    • 2% Non-fat powdered milk
    • 0.05% Tween
  3. Add appropriate amount of antibody for dilution (1:5,000, 1:1,000, 1:500, etc.).
  4. Prepare plastic bag:
    • Cut plastic tube to size of gel.
    • Seal bag with bag sealer set on 3.
    • Put membrane blot in bag.
    • Seal three sides of the plastic bag and cut excess (leave extra room on the top for when adding the antibody).
  5. Add antibody with pipette and aide.
  6. Completely wet membrane.
  7. Use a kimwipe to push out all the air bubbles.
  8. Seal bag closed once all bubbles are removed and cut bag excess.
  9. Put blot on rocker 1-2 hours at room temperature or overnight at 4 degrees Celsius.
  10. Take blot out of the bag and put in TBST rinse.
  11. TBST (For 1 Liter):
    • 900mls H2O
    • 100mls 10X TBS
    • 0.5mls Tween
  12. Save primary antibody. Store at 4 degrees Celsius.
  13. Put blot in tray with 100-200mls TBST and put on rocker for 3-5 minutes.
  14. Repeat rinse in TBST.
  15. Prepare secondary antibody (1:5,000):
    • 25mls TBST
    • 5ul secondary antibody
  16. Pour secondary antibody onto blot and put on rocker for at least 1 hour.
  17. Wash blot with 3 washes of TBST for 3-5 minutes each.
  18. Prepare Western Blotting Detection Solution:
    • 3mls solution A
    • 3mls solution B
  19. Lay glass plate down on bench and lay blot on glass plate.
  20. Put developer drop-wise over blots surface.
  21. Tilt blot to make sure the whole blot is covered.
  22. Let sit 1 minute.
  23. Lay out a piece of smooth seran wrap.
  24. Take blot off plate and let developer drip off.
  25. Lay blot protein side down onto seran wrap and fold seran wrap to seal blot in bag.
  26. Tape blot into cassette.
  27. Take Biomax light film, cassette, timer and scissors down to the dark room.
  28. Cut film into 2 pieces (fold upper right corner).
  29. Put film over blot and close cassette.
  30. Expose blot for 1 minute.
  31. Put film in developer.
  32. Expose blot for longer or shorter times depending on first exposure.
  33. Label film using blot (when it is still taped into the cassette).
  34. If using another antibody strip blot.