Team:Calgary/Notebook/Protocols/Process7

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Revision as of 02:19, 28 September 2011


Chloroplast isolation

This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.


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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