Team:SJTU-BioX-Shanghai/Protocols

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Project introduction. Inspired by the natural regulator of circadian bioclock exhibited in most eukaryotic organisms, our team has designed an E.coli-based genetic network with the toxin-antitoxin system so that the bacterium oscillates between two states of dormancy and activity (more...)

Protocols

Contents

General protocols

Standard biobrick preparation

  1. Centrifuge the distribution plates for a while so that the dry DNA precipitates onto the bottom of the wells;
  2. Penetrate the aluminum foil with the tip of a pipette (Caution: avoid damaging other wells);
  3. Add 15μl double-distilled water (ddH2O);
  4. Take 1~2μl to perform following operations.

Transduction

  1. Set the water bath to 42℃;
  2. Mark and place an EP tube of E.coli competent cells (ca.100μl) in a freezing box;
  3. Add 1~2μl plasmid solution and leave the tube in the freezing box for 30min;
  4. Heat shock the cells in the water bath for an exact time of 90s;
  5. Take out the tube and place it in the ice box for another 5min;
  6. Add 400μl (or 1000μl) of liquid medium into the tube and mark it;
  7. Shaking cultivate at 37℃ for 0.5~2h; meanwhile prepare the culture plates with corresponding antibiotic in the medium;
  8. Inoculate and cultivate the plates upside-down in a 37℃ incubator for 12~14h.

E.coli culture

  1. Add antibiotics of 0.1% concentration into a culture tube with 4~5ml liquid medium inside;
  2. Pick a single colony with a pipette tip from a culture plate and inoculate it into the liquid medium;
  3. Shaking culture in a 37℃ incubator overnight (ca. 12~16h).

Bacterium strain storage

  1. Prepare a 1.5ml EP tube and mark it;
  2. Add 400μl of 80% glycerin into the tube;
  3. Add 600μl of cultivated liquid medium into the tube;
  4. Store the tube in a -80C℃ fridge.

Agarose gel electrophoresis

  1. Gel preparation:
    • Ingredients:
      • (for a smaller piece of gel) 0.3g of agarose + 30ml of TBE + 4μl of EB;
      • (for a larger piece of gel) 0.8g of agarose + 80ml of TBE + 4μl of EB;
    1. Weigh out the required amount of agarose powder onto a piece of weighing paper;
    2. Add the agarose powder into a glass bottle specific for gel preparation;
    3. Add the required amount of TBE buffer into the bottle;
    4. Microwave the liquid with the bottle lid slightly open until it boils, so that the agarose powder gets well dissolved; meanwhile prepare the gel plates and ensure the right comb is fixed;
    5. Cool the bottle in a sink;
    6. When the solution feels warm but not hot (ca.60℃), add the required amount of EB with a pipette specific for gel preparation into the bottle;
    7. Shake the bottle carefully so that the EB is well mixed;
    8. Pour the liquid onto the gel plate;
    9. Wait for the liquid to be cured.
  2. Spotting:
    1. Place a disposable glove on the experiment table;
    2. Pipette correct amounts of loading buffer (so that the buffer will be diluted to a correct concentration) onto the glove to form a lane of buffer drops;
    3. Pipette correct amounts of samples into the buffer drops and mix them well;
    4. Pipette the correct DNA ladder (or marker) into a gel lane, usually the first or the middle-most one;
    5. Pipette the mixed samples into lanes of the gel; watch out for possible leakage which occurs when the pipette tip gets too deep and penetrates through the gel lane.
  3. Electrophoresis:
    • Voltage selection:
      • (for a general resolution) 160V;
      • (for a high resolution) 120V (and correspondingly longer electrophoresis time);
    1. After spotting the samples, install the lid of electrophoresis chamber and switch the output on;
    2. When the fastest lane runs to the center (or 2/3 the length if follow-up gel purification is intended) of the gel, switch off the output and transfer the gel into the UVP system;
    3. Power on UVP and observe the gel; take photos when necessary;
    4. Switch off the system and discard the gel into a special rubbish bin.

Double digestion

  • System selection:
    • For identification only, choose the 20μl system;
    • For following ligation, choose the 100μl system;
  • Refer to the Takara inventory for the correct buffer. Take EcoRI and PstI for an example:
  • 20μl system:
Plasmid(100ng/μl)10μl
EcoRI0.5μl
PstI0.5μl
1*H2μl
ddH2O7μl
  • 100μl system:
Plasmid(100ng/μl)30μl
EcoRI3μl
PstI3μl
1*H10μl
ddH2O54μl
  1. Prepare the system on a freezing box;
  2. Place the system under 37℃ temperature for 2~3h (for identification) or 9~12h (for ligation).

Tips for digestion

  1. Better limit the volume of restriction enzymes to less than 1/10 of the whole system, for the star activity would be enhanced under high concentration conditions. In most cases it is about 1/20 of the whole system.
  2. To avoid the case in which DNA concentration is too low and hence electrophoresis yields no result, ensure a minimum DNA amount of 50ng.
  3. For identification only, an estimated amount of 0.5μl of enzyme (8~20U) should correspond to 1μg of DNA; For ligation, 1μl of enzyme to 1μg of DNA.
  4. For identification only, a digestion time of 2~3h is enough; for ligation it’s better to leave the system overnight, otherwise massive false positive colonies would occur during the following steps of transduction and culture.

Special protocols

SuperScript™ III First-Strand Synthesis System for RT-PCR

The following procedure is designed to convert 1 pg to 5 μg of total RN or 1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA.
  1. Mix and briefly centrifuge each component before use.
  2. Combine the following in a 0.2- or 0.5-ml tube:
    Component Amount
    up to 5 μg total RNAn μl
    Primer: 50 μM oligo(dT)201 μl
    10 mM dNTP mix1 μl
    DEPC-treated waterto 10 μl
  3. Incubate at 65°C for 5 min, then place on ice for at least 1 min.
  4. Prepare the following cDNA Synthesis Mix, adding each component in the indicated order.
    Component 1 Rxn 10 Rxns
    10X RT buffer2 μl20 μl
    25 mM MgCl24 μl40 μl
    0.1 M DTT2 μl20 μl
    RNaseOUT (40 U/μl)1 μl10 μl
    SuperScript? III RT (200 U/μl)1 μl10 μl
  5. Add 10 μl of cDNA Synthesis Mix to each RNA/primer mixture, mix gently, and collect by brief centrifugation. Incubate as follows.
    Oligo(dT)20 or GSP primed:50 min at 50°C
    Random hexamer primed:10 min at 25°C, followed by 50 min at 50°C
  6. Terminate the reactions at 85°C for 5 min. Chill on ice.
  7. Collect the reactions by brief centrifugation. Add 1 μl of RNase H to each tube and incubate for 20 min at 37°C.
  8. cDNA synthesis reaction can be stored at -20°C or used for PCR immediately.

RNA extraction

  1. Get 1ml bacteria and centrifuge 1000rpm for 5mins, discard the pellet, rewash the cells with 1*PBS.
  2. Add in 800ul Trizol. nature at room temp for 5~10 mins. In this process, beat upon the cells with liquid-transfering gun will benefit its homogenization.
  3. The reagent for 12/24-well system
    Types of plate 35-cm 12-well 24-well Ratio by volume
    Trizol added 1ml 800ul 500ul Trizol: Chloro = 5:1
    Trizol: Isopro = 2:1
    Trizol: Ethanol = 1:1
    Chloroform added200ul160ul100ul
    Isopropyl-alcohol added500ul400ul250ul
    75% ethanol1ml800~ul500ul
  4. Add in the corresponding Chloroform, Vortexing 15s, quiescence at room temp for 2~3 mins.
  5. Centrifuge 12000g for 15 mins, recommended at 4~8℃, the liquid is separated in aqueous phase, red phenol-chloroform phase and an interfase. RNA is soluted in aqueous phase, 60% of the Trizol volume
  6. Transfer the aqueous phase carefully into a fresh EP tube. The remaining red phase can be utilized in the following DNA/protein extraction. Add in corresponding volume of Isopropyl-alcohol in accordance with the upper form, after vortexing, quiescence at room temp for 10 mins.
  7. Centrifuge at 12000g for 10 mins, recommended at 4~8 ℃. Now RNA are at the bottom of the tube.
  8. Discard the supernatant carefully, put in corresponding volume of 75% ethanol, vortexing 15s and centrifuge 7500g for 5 mins.
  9. Discard the supernatant carefully, place upside down to airing for 20~30 mins, in case of over airing and harm the RNA solution.
  10. Add in 40~50 ul DEPC to solute RNA, vortexing slightly and centrifuge. Set at 55~65℃ for 10 mins.
  11. Store at –70 ℃.
  12. The long-term storage: 75% ethanol, -20~-70℃ for about a year.
Preparation of reagents
  1. 1*PBS
  2. Trizol
  3. Sterilized EP tube (1.5/2.0ml format)
  4. Precooled 75% ethanol
  5. Isopropyl-alcohol
  6. Chloroform
  7. DEPC solution

Western blot

  1. Prepare gels for SDS-PAGE
  2. heat the sample buffer in heating water bath at 98°C
  3. boil every 15ul sample with 5ul sample buffer at 98 ℃ for 5 mins, marker 4 ul
  4. add in running buffer
  5. run until the marker goes to the end. (100 V constant voltage, 25 min; 200V, 40 min)
  6. SDS-PAGE Transfer Buffer:
    Tris-Base25 mM
    Glycin192 mM
    Methanol20%
    SDS1/1000
    For 1 L of buffer mix 3.03 g of Tris-Base, 14.4g of glycin and 200 mL of methanol; Bring to 1L with deionized water. Do not pH.
  7. Set up transfer from the gel to a filter paper in transfer buffer. The PVDF membrane should be as large as the gel. Keep the gel in transfer buffer in case of dry.
  8. Open the transfer chamber, put 2 layers of cushion to absorb water, 1 layer of filter paper from the transfer buffer. Put the gel carefully on the filter paper, and then add the PVDF membrane, add another filter paper and cushion.
  9. Add running buffer, run at 4℃, 350 mA constant current, for 65 mins
  10. Washing buffer:
    TBS500ml
    Tween200ul
  11. Blotting buffer:
    milk powder2.5g
    washing buffer50ml
  12. After electrophoresis, put the PVDF membrane into washing buffer, and then put in blotting buffer at 4 ℃, shaking overnight.
  13. Wash the membrane in washing buffer.
  14. Add primary antibody at proper dilution. Incubate the membrane for 2 hr with shaking at room temp.
  15. Wash the PVDH membrane for 3 times, 5 mins each.
  16. Add secondary antibody at proper dilution. Incubate the membrane for 2 hr with shaking at room temp.
  17. Wash the PVDH membrane for 3 times, 5 mins each.
  18. Detection by glimmering substrate, mix A and B solution by same volume.
  19. Transfer to a dark room.
  20. Put a preservative film on the table, take out the PVDH membrane, absorb the water with absorbant paper. Put the PVDF membrane on the glimmering substrate for 3 mins.
  21. After 3 mins, wrap the PVDF membrane with preservative film, put the film on PVDF membrane in the film cassette. React for 10 mins.
  22. Put in the development solution, wash by water, put in fixing solution, and wash by water again.
  23. PVDF membrane can be regenerated by TBS washing

QuikChange® II XL Site-Directed Mutagenesis Kit & QuikSolution™ Reagent

Enhanced amplification efficiencies 2.5–3.5μl QuikSolution/50μl reaction, with 3μl being optimal for most targets.
Mutant Strand Synthesis Reaction (Thermal Cycling)
Ensure that the plasmid DNA template is isolated from a dam+ E. coli strain. recommends using thin-walled tubes
  1. Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence. Purify these oligonucleotide primers prior to use in the following steps (see Mutagenic Primer Design).
  2. Prepare the control reaction as indicated below:
    • 5 μl of 10× reaction buffer
    • 2 μl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/μl)
    • 1.25 μl (125 ng) of oligonucleotide control primer #1 [34-mer (100 ng/μl)]
    • 1.25 μl (125 ng) of oligonucleotide control primer #2 [34-mer (100 ng/μl)]
    • 1 μl of dNTP mix
    • 3 μl of QuikSolution reagent
    • 36.5 μl of double-distilled water (ddH2O) to a final volume of 50 μl
    • Then add 1 μl of PfuUltra HF DNA polymerase (2.5 U/μl)
  3. Prepare the sample reaction(s) as indicated below:
    Stratagene recommends setting up an initial sample reaction using 10 ng of dsDNA template.
    • 5 μl of 10× reaction buffer
    • X μl (10 ng) of dsDNA template
    • X μl (125 ng) of oligonucleotide primer #1
    • X μl (125 ng) of oligonucleotide primer #2
    • 1 μl of dNTP mix
    • 3 μl of QuikSolution
    • ddH2O to a final volume of 50 μl
    • Then add 1 μl of PfuUltra HF DNA polymerase (2.5 U/μl)
  4. If the thermal cycler to be used does not have a hot-top assembly, overlay each reaction with ~30 μl of mineral oil.
  5. Cycle each reaction using the cycling parameters outlined in Table I. (For the control reaction, use a 5-minute extension time and run the reaction for 12 cycles.)
  6. Following temperature cycling, place the reaction tubes on ice for 2 minutes to cool the reactions to ≤37°C.
Cycling Parameters for the QuikChange® II XL Method
segment cycles temperature time
1195℃1 min
2 18 95℃ 50 sec
60℃50 sec
68℃1 minute/kb of
plasmid length
3168℃7 min
Dpn I Digestion of the Amplification Products
  1. Gently thaw the XL10-Gold ultracompetent cells on ice. For each control and sample reaction to be transformed, aliquot 45 μl of the ultracompetent cells to a prechilled Falcon? 2059 polypropylene tube.
  2. Add 2 μl of the β-ME mix provided with the kit to the 45 μl of cells. (Stratagene cannot guarantee highest efficiencies with β-ME from other sources.)
  3. Swirl the contents of the tube gently. Incubate the cells on ice for 10 minutes, swirling gently every 2 minutes.
  4. Transfer 2 μl of the Dpn I-treated DNA from each control and sample reaction to separate aliquots of the ultracompetent cells.
    As an optional control, verify the transformation efficiency of the XL10-Gold ultracompetent cells by adding 1 μl of 0.01 ng/μl pUC18 control plasmid (dilute the control provided 1:10 in high-quality water) to another 45-μl aliquot of cells.
    Swirl the transformation reactions gently to mix and incubate the reactions on ice for 30 minutes.
  5. Preheat NZY+ broth (see Preparation of Media and Reagents) in a 42°C water bath for use in step 8.
  6. Heat-pulse the tubes in a 42°C water bath for 30 seconds. The duration of the heat pulse is critical for obtaining the highest efficiencies. Do not exceed 42°C.
  7. Incubate the tubes on ice for 2 minutes.
  8. Add 0.5 ml of preheated (42°C) NZY+ broth to each tube, then incubate the tubes at 37°C for 1 hour with shaking at 225–250 rpm.
  9. Plate the appropriate volume of each transformation reaction, as indicated in the table below, on agar plates containing the appropriate antibiotic for the plasmid vector.
    For the mutagenesis and transformation controls, spread cells on LB–ampicillin agar plates containing 80 μg/ml X-gal and 20 mM IPTG (see Preparing the Agar Plates for Color Screening).
    Transformation reaction plating volumes
    Reaction Type Volume to Plate
    pWhitescript mutagenesis control250 μl
    pUC18 transformation control5 μl (in 200 μl of NZY+ broth)
    Sample mutagenesis250 μl on each of two plates
    (entire transformation reaction)
  10. Incubate the transformation plates at 37°C for >16 hours.

Annealing

Add 10μl forward, 10μl reverse primer into a tube, together with 80μl anneal buffer. Use PCR to cool down the temperature from 94℃ to 4℃ at the rate of 1℃/min.
DNA purification by phenol/chloroform method
Add 50μl obliges, 150μl ddH2O and 200μl phenol/chloroform into a tube. And spin at the speed of 12000rpm for 10 minutes. Extract the upper layer of the solution, added to a solution containing 400μl NaAc and 800μl absolute ethanol. Spin at the speed of 12000rpm for 10 minutes. Drop the upper layer of the solution. Add 500μl 70% ethanol to it. Spin at the speed of 12000rpm for 5 minutes. Drop the upper layer of the solution. Dry it and add 15μl ddH2O.

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