Team:Calgary/Notebook/Protocols/Process7
From 2011.igem.org
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
Isolation Buffer (500mL)
Equipment
Protocol
- 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.
- Harvest the cells (0.6-1.0 * 10^7 cells/mL) by centrifuging at 3000g for 10 min at 4°C.
- Wash the pellet with 100mL of 50mM HEPES-KOH (pH 7.5) by mixing and centrifuging at 3000g for 5 min at 4°C.
- Resuspend the pellet in 2mL of 50mM HEPES-KOH (pH 7.5) and hold at 4°C.
- Measure the chlorophyll concentration of the cells by a spectrophotometer.
- Dilute aliquots of cells with 3-0.3mg chlorophyll/mL using isolation buffer with 1% (w/v) BSA.
- 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).
- Collect whole cells and crude chloroplasts by centrifugation at 750g for 2 min at 4°C.
- Resuspend the pellet in 2mL isolation buffer using a paintbrush to prevent disruption of chloroplasts (smooth out any clumps).
- Overlay the suspenion on top of the gradients and centrifuge at 4200g for 15min at 4°C, the chloroplasts are at 45-65% interface.
- Collect the chloroplast band using a disposable glass pipet, dilute with 10mL isolation buffer with 0.1% (w/v) BSA.
- Centriduge at 670g for 1 min at 4°C to collect the organelles to remove Percoll.
- Resuspend the pellet gently using the paintbrush in 250 µL of 50mM HEPES-KOH pH 8.0 with 0.3M sorbitol.