Team:Lethbridge/Notebook/Protocols

From 2010.igem.org

(Difference between revisions)
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Back to [[Team:Lethbridge/Notebook|Notebook]]
Back to [[Team:Lethbridge/Notebook|Notebook]]
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=Common Protocols:=
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=<font color="white">Common Protocols:=
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==Competent Cell Transformation==
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==<font color="white">Competent Cell Transformation==
<ol>
<ol>
<li>Thaw 20&micro;L of aliquotted cells (DH5&alpha; or BL21(DE3)) on ice.</li>
<li>Thaw 20&micro;L of aliquotted cells (DH5&alpha; or BL21(DE3)) on ice.</li>
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</ol><br>
</ol><br>
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==Boiling Lysis Plasmid Preparation (Miniprep)==
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==<font color="white">Boiling Lysis Plasmid Preparation (Miniprep)==
<ol>
<ol>
<li>Aseptically transfer 1.5mL of each overnight culture to a 1.5mL microcentrifuge tube (MCT) and pellet the cells by centrifugation in a benchtop microcentrifuge (2min at 13000RPM)</li>
<li>Aseptically transfer 1.5mL of each overnight culture to a 1.5mL microcentrifuge tube (MCT) and pellet the cells by centrifugation in a benchtop microcentrifuge (2min at 13000RPM)</li>
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<li>Add 50&micro;L of TE (pH 8.0) containing RNase A and resuspend the plasmid DNA by flicking the base of the MCT with your finger. The plasmid DNA is ready for use or can be stored long term at -20<sup>o</sup>C.</ol>
<li>Add 50&micro;L of TE (pH 8.0) containing RNase A and resuspend the plasmid DNA by flicking the base of the MCT with your finger. The plasmid DNA is ready for use or can be stored long term at -20<sup>o</sup>C.</ol>
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==Restriction of Plasmid DNA (pDNA)==
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==<font color="white">Restriction of Plasmid DNA (pDNA)==
<ol>
<ol>
<li>In a 1.5mL microcentrifuge tube, add MilliQ H<sub>2</sub>O to final volume of 20&micro;L, 2&micro;L of restriction enzyme buffer, 2&micro;L of plasmid DNA, and 0.25&micro;L of each restriction enxyme.</li>
<li>In a 1.5mL microcentrifuge tube, add MilliQ H<sub>2</sub>O to final volume of 20&micro;L, 2&micro;L of restriction enzyme buffer, 2&micro;L of plasmid DNA, and 0.25&micro;L of each restriction enxyme.</li>
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</ol>
</ol>
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==Ligation of BioBricks==
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==<font color="white">Ligation of BioBricks==
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==Overexpression==
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==<font color="white">Overexpression==
<ol>
<ol>
<li>Inoculate two 5mL overnight cultures in LB media containing the appropriate antibiotic corresponding to the resistance of the plasmid backbone on which the gene is located; incubated at at 37<sup>o</sup>C in shaker.</li>
<li>Inoculate two 5mL overnight cultures in LB media containing the appropriate antibiotic corresponding to the resistance of the plasmid backbone on which the gene is located; incubated at at 37<sup>o</sup>C in shaker.</li>
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</ol>
</ol>
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==Maxiprep==
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==<font color="white">Maxiprep==
<ol>
<ol>
<li>Grow up a 500mL overnight culture in LB media containing the appropriate antibiotic corresponding to the restance of the plasmid.</li>
<li>Grow up a 500mL overnight culture in LB media containing the appropriate antibiotic corresponding to the restance of the plasmid.</li>

Revision as of 21:18, 19 September 2010




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Contents

Common Protocols:

Competent Cell Transformation

  1. Thaw 20µL of aliquotted cells (DH5α or BL21(DE3)) on ice.
  2. Gently pipet 2.0µL of DNA into competent cells
    ATTENTION:
    Do not perform any additional mixing
    Never use more DNA that 10% of the volume of the competent cells otherwise the cells get destroyed by osmotic shock
  3. Incubate the cells on ice for 30 minutes.
  4. Heat shock the cells in a water bath at 42oC for EXACTLY 45 seconds.
  5. Incubate the cells on ice for 5 minutes.
  6. Add 250µL sterile media to the cells and incubate at 37oC for 1 hour with shaking (200RPM).
  7. Plate 100µL and 50µL on prewarmed LB agar plate containing the appropriate antibiotic.
    For ligations, plate all 250µL.
  8. Leave plate for 10-15 minutes to soak the cell suspension into the agar.
  9. Flip plate over (agar on top)
  10. Incubate the plates in the 37oC incubator overnight

Boiling Lysis Plasmid Preparation (Miniprep)

  1. Aseptically transfer 1.5mL of each overnight culture to a 1.5mL microcentrifuge tube (MCT) and pellet the cells by centrifugation in a benchtop microcentrifuge (2min at 13000RPM)
  2. Remove and discard as much of the supernatant as possible by aspiration (e.g with a Pasteur Pipette). Do not suck up the cell pellet!!
  3. Rinse the cell pellet by washing 1.0mL of sterile MilliQ H2O gently down the inside wall of the MCT. This removes any traces of the supernatant adhering to the MCT wall while minimizing the disturbance to the cell pellet. (/li>
  4. Resuspend the cell pellet in 350µL of STET.
  5. Add 25µL of the Lysozyme solution and mix by inversion.
  6. Place the MCT in the bioling water bath for EXACTLY 35 seconds, remove and incubate on ice for 5 minutes.
  7. Pellet the cellular debris by centrifugation at 13000RPM for 15 minutes. Transfer the supernatant to a fresh MCT and discard the pellet.
  8. Precipitate the plasmid DNA by adding 40µL of 3.0M sodium acetate (pH 5.2) and 420µL isopropanol. Mix by inversion. Mix by inversion and incubate for 5 minutes at room temperature.
  9. Pellet the plasmid DNA by centrifugation at 13000RPM for 10 minutes at 4oC. A pellet of plasmid DNA should be visible at the base of the MCT when complete.
  10. Being careful not to disturb the pellet, discard the supernatant and rinse the pellet with 500µL of ice cold ethanol.
  11. Repeat above step.
  12. Invert and tap the open MCT several times against a piece of paper towel on your bench to remove as much ethanol as possible.
  13. Store the open MCT at room temperature for approximately 10 minutes to allow all remaining traces of ethanol to evaporate
  14. Add 50µL of TE (pH 8.0) containing RNase A and resuspend the plasmid DNA by flicking the base of the MCT with your finger. The plasmid DNA is ready for use or can be stored long term at -20oC.

Restriction of Plasmid DNA (pDNA)

  1. In a 1.5mL microcentrifuge tube, add MilliQ H2O to final volume of 20µL, 2µL of restriction enzyme buffer, 2µL of plasmid DNA, and 0.25µL of each restriction enxyme.
  2. Incubate at 37oC for 1 hour.
  3. Heat kill restriction enzymes on a heat block at 80oC for 20 minutes.
  4. Store short-term on ice or long-term at -20oC.

Ligation of BioBricks

Overexpression

  1. Inoculate two 5mL overnight cultures in LB media containing the appropriate antibiotic corresponding to the resistance of the plasmid backbone on which the gene is located; incubated at at 37oC in shaker.
  2. Transfer the two 5mL cultures into an 2000mL Erlenmeyer flask containing 500mL of LB media containing the same antibiotic.
  3. Measure and record initial read OD600 reading (Time = 0) blanking against the unincoulated LB media.
  4. Begin incubation at 37oC in shaker.
  5. Measure and record another OD600 reading 1 hour after (Time = 1) and continue record OD600 every 30 minutes (T + 0.5) until OD600 = 0.600 is reached.
  6. When OD600 has reached 0.600, aliquot out a 2mL sample and induce the 500mL culture (ie. if promoter is pLacI, induce with IPTG) and continue incubating in 37oC shaker.
  7. Aliquot 2mL samples and record the OD600 reading every hour for 3 hours following induction.
  8. To run SDS PAGE, centrifuge the samples at 13,000 rpm for 5 minutes and discard the supernatant. Resuspend the cell pellets with 100µL of 8M urea. Mix 10µL of the urea/cell sample with 5µL of 6X loading dye.

Maxiprep

  1. Grow up a 500mL overnight culture in LB media containing the appropriate antibiotic corresponding to the restance of the plasmid.
  2. Centrifuge the cells at 5000 rpm for 10 minutes, discard the supernatant, transfer pellet to a 50mL falcon tube, record the weight of the pellet, and store at -20oC. ( 1- 2.5g cell pellets are expected)
  3. Resuspend the cell pellet in 6mL Alkaline Lysis Soloution I (ALS1). Vortex carefully and slowly; may use a clean glass stir rod. Add 20&mirco;L of 1mg/mL RNase A.
  4. Add 1 mL of 10 mg/mL lysozyme (in 20mM Tris-HCl, pH 8.0)
  5. Incubate at room temperature for 10 minutes.
  6. Add 12mL of fresh ALS2, mix well, do not vortex, and incubate on ice for 10 minutes.
  7. Add 9mL of iceo cold ALS3, mix well, and incubate for 10 minutes on ice.
  8. Centrifuge at 4oC and 5000 rpm for 15 minutes.
  9. Decant supernatant filter within funnel into fresh 50mL centrigue tube.
  10. 1:1 phenol:chloroform extraction: In the fume hood, add 4mL of phenol/chloroform (1:1 -- 2mL of phenol + 2mL of chloroform), vortex for 15 seconds, centrifuge at 4000 rpm and 12oC for 4 minutes, and collect the upper aqueous phase. To the aqueous phase, add 4mL of chloroform, vortex for 15 seconds, centrifuge at 4000 rpm and 12oC for 4 minutes, and save the upper aqueous layer.
  11. Add 0.6 volumes of isopropanol to the saved aqueous layer and incubate on ice for 10 minutes. (Alternatively, precipitate overnight at -20oC)
  12. Centrifuge at 4oC and 5000 rpm for 15 minutes.
  13. Decant supernantant into a fresh falcon tube (can be saved for further plasmid DNA isolation)
  14. Wash DNA pellet with 2mL 70% ethanol; centrifuge at 4oC and 5000 rpm for 5 minutes.
  15. Air dry the DNA pellet; resuspend in 4mL 20mM Tris-HCl pH 8.0 by vortexing.
  16. Add 200µL 1mg/mL RNase A and incubate at room temperature overnight.
  17. Ethanol precipitation: Add 0.1 volumes of 3M Na-acetate pH 5.2, add 2 volumes of cold ethanol (-20oC), incubate 30 minutes on ice (or longer at -20oC).
  18. Centrifuge at 4oC and 4500 rpm for 15 minutes; carefully remove supernatant with 200µL pipette.
  19. Wash pellet with 750µL of 70% ethanol, centrifuge at 4oC and 4500 rpm for 2 minutes, remove supernatant, and air dry the pellet for 10 minutes.
  20. Resuspend the pellet in 200µL of MilliQ H2O (or TE).

Solutions:
ALS1: 50mM glucose, 25mM Tris-Cl, 10mM EDTA pH 8.0
ALS2: 0.2M NaOH, 1% (w/v) SDS
ALS3: 60mL of 5M K-acetate, 11.5mL glacial acetic acid, 28.5mL MilliQ H2O
RNase A: 1mg/mL in 20mM Tris-HCl, pH 8.0 Lysozyme: 10mg/mL in 20mM Tris-HCl, pH 8.0

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