Team:OUC-China/Note Book/Protocol

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Protocol

    Elementary Operation

  • Gel Extraction Kit

    • HiBind ® DNA Mini Columns
    2 ml Collection Tubes
    Binding Buffer* (XP2)
    Elution Buffer
    SPW Wash Buffer Concentrate

    1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended however, that fresh TAE buffer, or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.

    2. When adequate separation of bands has occurred, CAREFULLY excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

    3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 ml microcentrifuge tube. Assuming a density of 1g/ml of gel, the volume of gel is derived as follows: a gel slice of mass 0.3g will have a volume of 0.3 ml. Add an equal volume of Binding Buffer (XP2). Incubate the mixture at 55°C -60°C for 7 min or until the gel has completely melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes.

    4. Place a HiBind DNA Mini Column in a provided 2 ® ml collection tube.

    5. Apply 700μl of the DNA/agarose solution to the HiBind® DNA Mini Column, and centrifuge at 10, 000 x g for 1 min at room temperature.

    6. Discard liquid and place the HiBind® DNA Mini Column back into the same collection tube. For volumes greater than 700 μl, load the column and centrifuge successively, 700 μl at a time. Each HiBind® DNA Mini Column has a total capacity of 25μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.

    7. Add 300μl of Binding Buffer (XP2) into the HiBind® DNA Mini Column. Centrifuge at 10,000 x g for 30 to 60 seconds at room temperature to wash the HiBind® DNA Mini Column. Discard the flowthrough and re-use the collection tube.

    8. Wash the HiBind® DNA Mini Column by adding 700μl of SPW Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000xg for 30 to 60 seconds at room temp.(NOTE: SPW Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, SPW Wash Buffer must be brought to room temperature before use.)

    9. OPTIONAL: repeat step 8 with another 700μl of SPW Wash Buffer diluted with absolute ethanol.(NOTE: Perform the second wash step for any salt sensitive downstream applications)

    10. Discard liquid and centrifuge the empty HiBind® DNA Mini Column for 2 min at maximal speed ( ≥ 13,000xg) to dry the column matrix.

    11.Do not skip this step, it is critical for the removal of ethanol from the HiBind® DNA column.

    12. Place a HiBind® DNA Mini Column into a clean 1.5ml microcentrifuge tube. Add 30-50μl (depending on desired concentration of final product) of Elution Buffer (10mM Tris-HCl, pH 8.5) directly onto the column matrix and incubate at room temperature for 1 minute. Centrifuge for 1 min at maximal speed (≥13,000xg) to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.(NOTE: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent of pH. If eluting DNA with deionized water, make sure that the pH is around 8.5.)

  • Double Digestion

    • 25μl system

    Enzyme both 0.5μl
    Buffer 2μl
    BSA 0.2μl
    ddH2O 1.8μl
    DNA 20μl

    50μL system

    Enzyme both 1μl
    Buffer 5μl
    BSA 0.5μl
    ddH2O 2.5μl
    DNA 40μl

    Procedure

    1.Check NEB instruction booklet to find out the best buffer for your double digest system.
    2.Add all of the above to a 250μl Eppendorf Tube.
    3.Put the tube in metal bath for 3.5h at 37°C .
    4.Put the tube in metal bath for 20min at 80°C to inactivate the enzymes.

  • Transformation

    • 1.Take competent cells out of the -80°C refrigerator and put it in ice.
    2. When the competent cells dissolve (about 10min), subpackage those cells into 1.5mL Eppendorf Tubes for 50μl per tube.
    3.Add 10μl DNA per tube, standing for 30min.
    4.Put the tubes into 42°C metal bath for about 80s.
    5.Put the 1.5ml tubes back in ice for 5min.
    6.Add 800μl LB fluid medium into the 1.5ml tubes and then shaking culture for 1h.
    7.Extract 100μl bacterium suspension, spread it on agar culture medium which has relevant antibiotic.

  • Conservation

    • 40％ Glycerinum
    Bacterium Suspension
    1.Add 800μl 40％ glycerinum and 800μl bacterium suspension to a 2ml Eppendorf Tube.
    2.After mixing well, put the tube into -20°C refrigerator.

  • Gibson Assembly

    • 5x Isothermal Reaction Mix

    3ml 1M Tris-Hcl (pH 7.5)
    300ml 1M MgCl2
    60ml 100 mM dGTP
    60ml 100 mM dATP
    60ml 100 mM dTTP
    60ml 100 mM dCTP
    300ml 1 M DTT
    1.5g PEG-8000
    300mL 100 mM NAD
    Balance ddH2O
    6ml Total

    Assembly Master Mix

    320 ml 5x Isothermal Reaction Mix
    0.64 ml 10 U/ml T5 exonuclease
    20 ml 2 U/ml Phusion® DNA Polymerase
    16 ml 40 U/ml Taq DNA Ligase
    Balance ddH2O
    1.2ml Total

    Procedure

    1. Amplify the interest fragments through PCR and purify the interest DNA fragments through electrophoresis.
    2. Extract 15μl assembly master mixture and add 5μl DNA in to the mixture. You’d better operate these on ice. What’s more, the different DNA fragments should be in equimolar amounts.
    3. Incubate the mixture at 50 °C for 15 to 60 min (60 min is optimal).
    4. Transform as usual.

  • Sterilization

    • 1. Check the water level and tightness to ensure the security and sterilizing effct of the Autoclaves Sterilizer.
    2. Push the start button and then switch on the air valve.
    3. Close the air valve when the temperature reaches to 100℃.
    4. Switch off the Autoclaves Sterilizers 0.5h after step 3.
    5. Switch on the air valve when the air pressure meter is 0.
    6. Open the pot when the temperature inside is acceptable.

    Attention:

    1. Packs before sterilization should be separated into two groups. One of them is clean and the other carries bacterium. In order to avoid cross contamination, those two should be sterilized separately.
    2. You‘d better use distilled water in the sterilizer for tap water which contains ion salts is adverse to the Autoclaves Sterilizer.

  • Luria-Bertani medium

    • Luria-Bertani liquid medium

    Tryptone 10g/L
    Yeast extract 5g/L
    NaCl 10g/L
    pH: 7.4

    Luria-Bertani solid medium

    Tryptone 10g/L
    Yeast extract 5g/L
    NaCl 10g/L
    Agar 15g/L
    pH: 7.4

  • Plasmid extraction

    • Materials Supplied by User:
    Micro-centrifuge capable of at least 10,000 x g
    Sterile 1.5 ml or 2 ml Centrifuge Tubes
    Sterile 10-20 ml Centrifuge Tubes
    Absolute (96%-100%) Ethanol
    Sterile 50 ml centrifuge tubes
    Centrifuge with swinging bucket rotor

    1. Isolate a single colony from a freshly streaked selective plate, and inoculate a starter culture of 1- 5 ml LB medium containing the appropriate selective antibiotic. Incubate for ~ 12-16 h at 37℃ 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. It is strongly recommended that an end. A negative strain of E.coli be used for routine plasmid isolation. Examples of such strains include DH5a and JM109.

    2. Pellet bacterial cells by centrifugation at 10,000 x g for 1 min at room temperature. Decant or aspirate medium and discard.

    3. Resuspend the bacterial pellet by adding 250ul of Solution I/RNase. A solution, and vorteing (or pipetting up and down). Transfer suspension into a new 1.5 ml micro-centrifuge tube. Complete resuspension (no visible cell clumps) of cell pellet is vital for obtaining good yields.

    4. Add 250ul of Solution II and gently mix by inverting and rotating tube several times to obtain a clear lysate. A 2 minute incubation may be necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 min. (Store Solution II tightly capped when not in use to avoid acidification of Solution II from CO2 in the air.)

    5. Add 350ul of Solution III and mix immediately by inverting several times until a flocculent white precipitate forms. It is vital that the solution is mixed thoroughly, and immediately after the addition of Solution III to avoid localized precipitation.

    6. Centrifuge at ≥13,000 x g for 10 minutes at room temperature. A compact white pellet will form. Promptly proceed to the next step.

    7. Prepare the column by adding 100ul of Equilibration Buffer placed in a 2 mL collection tube. Centrifuge at≥13,000 x g for 30-60 seconds. Discard the flow through liquid.

    8. Add the cleared supernatant by CAREFULLY aspirating it into a clean HiBind DNA Miniprep Column (I) assembled in a provided 2 ml collection tube. Ensure that the pellet is not disturbed and that no cellular debris has carried over into the column. Centrifuge for 1min at≥13,000 x g at room temperature to completely pass lysate through the HiBind DNA Miniprep Column (I).

    9. Discard flow -through liquid. Add 500ul of Buffer HB to w ash the HiBind DNA Miniprep Column (I). Centrifuge for 30 to 60 seconds≥13,000 x g at room temperature to completely pass liquid through the HiBind DNA Miniprep Column (I).This step ensures that residual protein contaminations are removed, thus ensuring high quality DNA that will be suitable for downstream applications.

    10. Discard flow -through liquid and add 700ul of DNA Wash Buffer diluted with absolute ethanol to w ash the HiBind DNA Miniprep Column (I). Centrifuge for 30 to 60 seconds≥13,000 x g at room temperature to completely pass liquid through the HiBind Miniprep Column (I) and discard flow -through liquid. NOTE: DNA Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, DNA Wash Buffer must be brought to room temperature before use.

    11. OPTIONAL: Repeat w ash step with another 700ul of DNA Wash Buffer diluted with absolute ethanol.

    12. Centrifuge the empty column for 2 min at≥ 13,000 x g to dry the column matrix. Do not skip this step, it is critical for good yields.

    13. Place the column into a clean 1.5 ml micro-centrifuge tube. Add 50ul to 100ul. (Depending on desired concentration of final product) of Elution Buffer (Tris-HCl, pH 8.5) or sterile deionized water directly onto the column matrix and centrifuge for 1 min at≥13,000 x g to elute DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

    14. Yield and quality of DNA: Determine the absorbance of an appropriate dilution of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows: DNA concentration = A 260 x 50 x (Dilution Factor) ug/ml. A ratio of (A 260) / (A 280) is an indication of nucleic acid purity. A value greater than 1.8 indicates > 90% nucleic acid purity. Alternatively, yield (as well as quality) can sometimes be best determined by agarose gel/ethidium bromide electrophoresis by comparison to DNA samples of known concentrations. Typically, the majority of the eluted DNA is in monomeric supercoil form, although concatamers may also be present.

  • PCR Product Purification

    • 1.Place PCR production in a provided 1.5ml collection tube, add Buffer p3 five times the volume of PCR production.(Note: If the volume of PCR production is less than 100μl，add ddH2O until the volume is 100μl)

    2.Add the mixture to a Spin Column, centrifuge for 30 seconds≥8000xg at room temperature to completely pass liquid through the Spin Column, discard flow-through liquid.

    3.Add 500ul of Wash Solution to wash Spin Column, centrifuge for 30 seconds≥9000 x g at room temperature to completely pass liquid through the Spin Column, discard flow -through liquid

    4.Repeat step 3.

    5.Place the column into a clean 1.5 ml micro-centrifuge tube. Add 15ul to 40ul.(Depending on desired concentration of final product) of Elution Buffer (Tris-HCl, pH 8.5) or sterile deionized water directly onto the column matrix incubate at room temperature for 1 minute, centrifuge for 1 min at≥13,000xg to elute DNA.

  • Ligation system

    • 1.0μl 10x T4 ligase buffer
    6:1 molar ratio of insert to vector (~10ng vector)
    Add (8.5 - vector and insert volume) μl ddH2O
    0.5μl T4 Ligase

    Calculating Insert Amount

    The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.

  • Inoculation

    • 1.Add 100μl relevant antibiotic to conical flask which contains 100mL Luria-Bertani liquid medium.
    2.Add 50-100μl bacterium suspension to conical flask.
    3.Put conical flask in incubator.

  • PCR

    • 25μl reaction system

    Taq enzyme： 0.15μl
    DNA or bacterium solution： 1.5μl
    Primer： both 0.5μl
    dNTP mix: 2μl
    10×taq buffer: 2.5μl
    ddH2O： 17.85μl

    50μl reaction system

    Taq enzyme： 0.3μl
    DNA or bacterium solution： 3μl
    Primer： both 1μl
    dNTP mix: 4μl
    10×taq buffer: 5μl
    ddH2O ： 37.5μl

    PCR parameter

    Step1. 95℃： 3min (If substrate is bacterium suspension, time is 10 min。)
    Step2. 95℃ 45s
    Step3. 5℃ below the Lower temperature of primers 2min
    Step4. 72℃ 1min per 1000bp
    GOTO step2 for 35times
    Step 5 72℃ three times longer than step 4

    Ferritin Protocol

  • Mineralization Scheme

  • Inoculation and Activation

    • For example:
    Add 5ml LB fluid medium, 20μl bacterial suspension and relevant antibiotics into 12ml tube. Cultivate for 10h.

  • Inoculation and cultivation

    • Parallel Options:

    Aspirate the bacterial suspension after activation and inoculate it into the new LB liquid medium until the OD value is 0.6~0.7 (about 3h).

  • Protein harvesting process and relevant parameters

    • Cultivation till OD600=0.6 37.0℃; 220rpm 3-4h
    Cultivation under induction 18.0℃; 220rpm 4h
    Cultivation with rich iron source 18.0℃; 220rpm overnight
    Centrifugation 3 times 4℃; 4000rpm 2h
    Resuspension by PBS after each centrifugation 0.5h
    Ultrasonication ice-water bath; 100-120W; 5s/10s; 12min per tube 2h
    Centrifugation 4℃; 8000rpm 0.5h

  • Induction and mineralization

    • pET’s final concentration of IPTG is 0.8mM and the the pQE’s final concentration is 1mM. Then add iron source making the final concentration of ferric ammonium citrate solution is 1 mM. Cultivate overnight.

    Note: The group without mineralization means it was cultivated in the same condition without adding iron source. Induction is in the same way.

  • Mag-attraction Test

    • Take out 12mL bacterium solution per erlenmeyer flask and centrifuge on 8,000g, then discarding the supernatant with PBS for 3 times to wash off the metal ions avoid false positive. Finally the bacterium is resuspended within 1mL PBS.

    Add the above-mentioned 1mL liquid into the disposable plate with 15mL PBS and 2mL LB liquid medium.

    Put a piece of black paper between the plate and the magnet. (It can be omitted if the background is black)

    Observe the bacterial formating phenomenon after 10h.

  • Ultrasonication

    • Centrifuge the rest of the bacterium solution (288ml/flask), and then add 10mlPBS every flask and resuspend 1~2 times.

    Use Ultrasonic Cell Disruptor whose power is 100W to disrupt bacterium, run 5s and then rest 10s, the total time is 3.5min.

    Centrifuge the bacterium after untrasonication, 13000rmp for 10min, gather the supernatant and sediment, respectively.

  • Inspect the rate of ultrasonication by microscopic examination

    • Pick up 20μl sample of ultrasonication, spread it out on the microslide. Move the microslide through alcohol lamp flame to fix the cell. Place a drop of crystal violet to cover the sample, staining 1min, wash the dye and dry it. Observe it under the microscope.

  • Purification and concentration

    • Pick up the supernatant of induction series and make it through Ni-NTA spin columns in order to purify the ferritin.

    Loading Buffer

    250mM NaCl
    PBS(1x): up to 1L

    Wash Buffer

    20mM Imidazole
    Loading Buffer: up to 1L

    Elution Buffer

    250mM Imidazole
    Loading Buffer: up to 1L

    Through Ni-NTA spin column

    Filtrate all the liquid in the tube.
    Add loading buffer whose volume is 10ml column volumes.
    After filtration, add supernatant, mix and filtrate well. (Repeat several times if necessary.)
    After filtration, add 10ml loading buffer to wash the volume wall.
    Add 20ml wash buffer and filtrate. (Repeat several times.)
    Add 10ml elution buffer, standing for 2min, and then capture the protein.
    Add 15ml elution buffer to wash off the residual protein.
    The next turn.
    The end of the purification, first use 6M guanidine hydrochlorideliquid to wash the Ni-NTA spin column then use water to restore for short time or ethyl alcohol for long time.

    Concentration

    Add the ferritin after purification into 50ml Millipore concentration tube , centrifuge in 4000g for 7min and capture the residue.

  • SDS PAGE

    • Preparation of gels

    Assemble gel caster with spacer and plate.
    The gel whose thickness is 0.75mm.( Table shows)

    13.2% and 5% are the rations of acrylamide.You needn't to add TEMED quantitatively, but don't over.

    Sample preparing

    Add 5μl 5×SDS Loading Buffer and 20μl ferritin sample (sediment is resuspended by PBS) into a 200ul PCR tube and mix sufficiently, heat at 100℃ about 5~10min.

    Centrifuge the sample.

    Sample loading

    20μl sample is loaded when the gel is 0.75mm.
    Except sediment and supernatant, loading a purified sample is necessary.
    Run at 110V for 1.5h.
    Dye the gel with R250 solution for 30min.
    Remove the R250 solution from the gel with destaining solution.

    SDS PAGE Buffer (10x) 1L

    Tris: 30.2g
    Glycine: 188g
    ddH2O : up to 1L

    SDS PAGE Buffer (1x) 500ml

    ddH2O : 450ml
    10X Protein Electrophoresis Buffer: 50ml

    SDS Loading Buffer (5x) 4ml

    ddH2O : 1.76ml
    1M Tris-HCl(pH6.8): 0.24ml
    Glycerinum: 1ml
    10%SDS: 0.8ml
    β-mercaptoethanol (BME): 0.2ml
    Finally, use tips to dip in bromophenol blue power and mix together with the liquid above.

    R250 Solution 1L

    R-250 Coomassie brilliant blue (CBB)：1g
    Ethyl alcohol: 250ml
    Glacial acetic acid: 100ml
    ddH2O : 650ml

    Destaining Solution 1L

    Ethyl alcohol: 100ml
    Glacial acetic acid: 100ml
    ddH2O : 800ml

  • Native PAGE

    • Preparation of gels

    Assemble gel caster with spacer and plate.
    The gel whose thickness is 0.75mm.( Table shows)

    Sample preparing and loading

    Loading Buffer: 10μl
    Ferritin Suspension: 40μl
    Load example 40μl when the gel is 1.5mm, while loading 20μl of sample when the gel is 0.75ml.
    Run at 110V for 3.5h.
    One of the gels is dyed by R250 solution (refer to SDS PAGE) and the other is dyed by potassium ferrocyanide liquid.

    Potassium Ferrocyanide Staining Solution(100ml)

    50mM Tris: 0.6057g
    100Mm NaCl: 0.5844g
    100mM Potassium Ferrocyanide: 4.2239g
    Using HCl aq to adjust the pH to 7.5.
    Cyanide is toxic, do wear mask during preparing.
    Dye 10~20min without light.

    Developing-Coloring Solution

    10% Trichloroacetic acid (TCA)
    10% Methyl alcohol (Add it into solution when using.)
    ddH2O : up to the volume that is needed
    Develop color for 30min.

    Destaining Solution

    5% Sulfosalicyl
    10% Trichloroacetic acid (TCA)
    Destain the gel until the dye is removed from the background.

    Native PAGE Buffer (1x) 500ml

    ddH2O : 450ml
    Protein electrophoresis buffer (10x): 50ml

    Native Loading Buffer (5X) 4ml

    ddH2O :1.76ml
    1M Tris-HCl (pH6.8): 0.24ml
    Glycerinum: 1ml
    β-mercaptoethanol (BME): 0.2ml
    Finally, use tips to dip in bromophenol blue power and mix together with the liquid above.