Team:Calgary/Notebook/Protocols/Process7

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TITLE=Plasmid Extraction|
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TITLE=Chloroplast Isolation|
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<p>
<p>
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This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.</p>
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This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR. The procedure is adapted from Mason, Bricker & Moroney <i>et al.</i> (2006) publication in Nature Protocols.</p>
<br>
<br>
<h4> Reagents </h4>
<h4> Reagents </h4>
-
<ol>
 
-
<li>LB broth, pH 7</li>
 
-
<li>10g tryptone</li>
 
-
<li>5g yeast extract</li>
 
-
<li>5g NaCl</li>
 
-
<li>20% sucrose (autoclaved)</li>
 
-
<li>Triton X-100</li>
 
-
<li>500mM EDTA stock (pH 8.0)</li>
 
-
<li>Tris-HCl 50mM</li>
 
-
<li>NaCl 3M</li>
 
-
<li>Isopropanol</li>
 
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<li>Autoclaved Milli-Q water</li>
 
-
</ol>
 
-
<br>
+
<li>HEPES (N-[2-hydroxyethyl] piperazine N'[2-ethane-sulfonic acid])</li>
-
<h4> Procedure </h4>
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<li>Sorbitol</li>
 +
<li>Percoll</li>
 +
<li>PEG 6000</li>
 +
<li>Ficoll</li>
 +
<li>Bovine serum albumin (BSA)</li>
 +
<li>Isoascorbic acid</li>
 +
<li>Sodium pyrophosphate</li>
 +
<li>Glutathione</li>
 +
<li>MgCl<sub>2</sub>.6H<sub>2</sub>O</li>
 +
<li>MnCl<sub>2</sub></li>
 +
<li>Sodium-EDTA</li>
 +
 +
<h4>Isolation Buffer (500mL)</h4>
 +
<li>27.4g of 300mM sorbitol</li>
 +
<li>5.96g of 50mM HEPES-KOH (pH 705)</li>
 +
<li>2mL 0.5M stock of 2mM Na-EDTA (pH 8.0)</li>
 +
<li>0.1g of 1mM MgCl<sub>2</sub>.6H<sub>2</sub>O</li>
 +
<li>5.0g of 1% BSA</li>
 +
 +
<h4> Equipment </h4>
 +
 +
<li>High-speed centrifuge and rotor</li>
 +
<li>Spectrophotometer for chlorophyll measurements</li>
 +
<li>Orbital shaker, light suitable for algal cell growth, and timer to control light settings</li>
 +
<li>27-gauge * 0.5 inch stainless stell syringe needles</li>
 +
<li>B-D Multifit 10mL glass Leuer-lock syringe</li>
 +
<li>15mL Corex centrifuge tubes</li>
 +
<li>Hemocytometer (cell counter)</li>
 +
<li>Fine (artist style) paint brush</li>
 +
<br></br>
 +
<p><h4>Protocol</h4></p>
<ol>
<ol>
-
<li>Inoculate a loopful of Pseudomonas sp. at 25oC, in 10mL LB broth (10g tryptone; 5g yeast extract, 5g NaCl, 1000mL distilled H2O, pH 7.0), and incubate for 16-18hr.</li>
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<li>Inoculate algal cells at a density of 4*10^4 cells/mL in 1L of minimal media. Grow cells under white light for 5 days.</li>
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<li>Centrifuge 1.5 ml of a 16-18hr bacterial culture for 1 min at 11,500 x g in a polypropylene centrifuge tube.</li>  
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<li>Harvest the cells (0.6-1.0 * 10^7 cells/mL) by centrifuging at 3000g for 10 min at 4°C.</li>
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<li>Remove supernatant by aspirating, leaving the pellet as dry as possible.</li>
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<li>Wash the pellet with 100mL of 50mM HEPES-KOH (pH 7.5) by mixing and centrifuging at 3000g for 5 min at 4°C.</li>
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<li>Add to each tube, 400µL of 8% sucrose, 5.0% Triton X-100, 50 mM EDTA, and 10mM Tris HCI (pH 8.0). Mix well.</li>
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<li>Resuspend the pellet in 2mL of 50mM HEPES-KOH (pH 7.5) and hold at 4°C.</li>
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<li>Add 50µL of freshly prepared lysozyme solution (10mg/mL in 10mM Tris HCl, pH8), mix by inverting 3X.  Lysozyme digests bacterial cell wall.</li>
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<li>Measure the chlorophyll concentration of the cells by a spectrophotometer.</li>
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<li>Immediately incubate at 100oC for 10, 20, 40, 80s.</li>
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<li>Dilute aliquots of cells with 3-0.3mg chlorophyll/mL using isolation buffer with 1% (w/v) BSA.</li>
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<li>Spin for 10min 11,500Xg at room temp.</li>
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<li>Quickly draw the diluted cells into the syringe, attach a 27-gauge needle, and pass through the needle at 0.1mL/s flow rate (~80psi).</li>
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<li>Remove pellet with sterile forceps.</li>
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<li>Collect whole cells and crude chloroplasts by centrifugation at 750g for 2 min at 4°C.</li>
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<li>Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.</li>
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<li>Resuspend the pellet in 2mL isolation buffer using a paintbrush to prevent disruption of chloroplasts (smooth out any clumps).</li>
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<li>Incubate 30min at -20oC.</li>
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<li>Overlay the suspenion on top of the gradients and centrifuge at 4200g for 15min at 4°C, the chloroplasts are at 45-65% interface.</li>
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<li>Centrifuge 15min for 11,500Xg at 4oC.</li>
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<li>Collect the chloroplast band using a disposable glass pipet, dilute with 10mL isolation buffer with 0.1% (w/v) BSA.</li>
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<li>Decant supernatant, invert and drain on clean paper towel.</li>
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<li>Centriduge at 670g for 1 min at 4°C to collect the organelles to remove Percoll.</li>
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<li>Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).</li>
+
<li>Resuspend the pellet gently using the paintbrush in 250 µL of 50mM HEPES-KOH pH 8.0 with 0.3M sorbitol.</li>
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<li>Incubate for 1hr at 4oC in dark. </li>
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<li>Run a small amount of this sample on gel electrophoresis on 0.7% (w/v0) agarose.  With the rest, submit to further purification.</li>
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</ol>
</ol>
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<br>
 
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<h4>Further purification (Plasmid from putida)</h4>
 
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<ol>
 
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<li>Layer the resuspended DNA on a 2.5mL bed of saturated CsCl in a polymer tube.  Centrifuge for 14hr at 14 000 rpm in a fixed-angle 30 rotor at 2°C.  After the run, ~25mL of liquid can be discarded from the top without disturbing the remainder.  Mix the lower part to form the concentrated lysate.</li>
 
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<li>Slowly dissolve ~5.7g CsCl to the concentrated lysate, until the refractive index is 1.399.  Mix solution with Syber-safe or gel-red.  Centrifuge for 40hr in a Spinco fixed-angle rotor 50 at 105 000 x g at 12°C.  After this run, 2 well-separated bands should be able to be seen.  Alternatively, do only this spin where DNA mixed with CsCl is concentrated to a refractive index of 1.399 with the fluorescent DNA stain.</li>
 
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<li>Because the DNA was infused with dye, 2 well-separated bands will appear.  The upper band is linear and non-circular DNA (junk).  The lower band is the plasmid of interest.  Remove the upper layer with a micropipette.  After it is removed, pool bands from several tubes centrifuge again SW50.1 rotor for 20h at 40 000rpm.  Again there will be 2 bands and the lower band is the desired intact plasmid.</li>
 
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Latest revision as of 04:13, 29 September 2011


Chloroplast Isolation

This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR. The procedure is adapted from Mason, Bricker & Moroney et al. (2006) publication in Nature Protocols.


Reagents

  • HEPES (N-[2-hydroxyethyl] piperazine N'[2-ethane-sulfonic acid])
  • Sorbitol
  • Percoll
  • PEG 6000
  • Ficoll
  • Bovine serum albumin (BSA)
  • Isoascorbic acid
  • Sodium pyrophosphate
  • Glutathione
  • MgCl2.6H2O
  • MnCl2
  • Sodium-EDTA

    • Isolation Buffer (500mL)

  • 27.4g of 300mM sorbitol
  • 5.96g of 50mM HEPES-KOH (pH 705)
  • 2mL 0.5M stock of 2mM Na-EDTA (pH 8.0)
  • 0.1g of 1mM MgCl2.6H2O
  • 5.0g of 1% BSA

    • Equipment

  • High-speed centrifuge and rotor
  • Spectrophotometer for chlorophyll measurements
  • Orbital shaker, light suitable for algal cell growth, and timer to control light settings
  • 27-gauge * 0.5 inch stainless stell syringe needles
  • B-D Multifit 10mL glass Leuer-lock syringe
  • 15mL Corex centrifuge tubes
  • Hemocytometer (cell counter)
  • Fine (artist style) paint brush


    • Protocol

    1. Inoculate algal cells at a density of 4*10^4 cells/mL in 1L of minimal media. Grow cells under white light for 5 days.
    2. Harvest the cells (0.6-1.0 * 10^7 cells/mL) by centrifuging at 3000g for 10 min at 4°C.
    3. Wash the pellet with 100mL of 50mM HEPES-KOH (pH 7.5) by mixing and centrifuging at 3000g for 5 min at 4°C.
    4. Resuspend the pellet in 2mL of 50mM HEPES-KOH (pH 7.5) and hold at 4°C.
    5. Measure the chlorophyll concentration of the cells by a spectrophotometer.
    6. Dilute aliquots of cells with 3-0.3mg chlorophyll/mL using isolation buffer with 1% (w/v) BSA.
    7. Quickly draw the diluted cells into the syringe, attach a 27-gauge needle, and pass through the needle at 0.1mL/s flow rate (~80psi).
    8. Collect whole cells and crude chloroplasts by centrifugation at 750g for 2 min at 4°C.
    9. Resuspend the pellet in 2mL isolation buffer using a paintbrush to prevent disruption of chloroplasts (smooth out any clumps).
    10. Overlay the suspenion on top of the gradients and centrifuge at 4200g for 15min at 4°C, the chloroplasts are at 45-65% interface.
    11. Collect the chloroplast band using a disposable glass pipet, dilute with 10mL isolation buffer with 0.1% (w/v) BSA.
    12. Centriduge at 670g for 1 min at 4°C to collect the organelles to remove Percoll.
    13. Resuspend the pellet gently using the paintbrush in 250 µL of 50mM HEPES-KOH pH 8.0 with 0.3M sorbitol.

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