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Team:Calgary/Notebook/Protocols/Process7
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| - | TITLE= | + | TITLE=Chloroplast Isolation| |
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<p> | <p> | ||
| - | This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.</p> | + | 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> | ||
| Line 19: | Line 19: | ||
<li>Sodium pyrophosphate</li> | <li>Sodium pyrophosphate</li> | ||
<li>Glutathione</li> | <li>Glutathione</li> | ||
| - | <li> | + | <li>MgCl<sub>2</sub>.6H<sub>2</sub>O</li> |
| - | <li> | + | <li>MnCl<sub>2</sub></li> |
<li>Sodium-EDTA</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> | <h4> Equipment </h4> | ||
| Line 33: | Line 41: | ||
<li>Hemocytometer (cell counter)</li> | <li>Hemocytometer (cell counter)</li> | ||
<li>Fine (artist style) paint brush</li> | <li>Fine (artist style) paint brush</li> | ||
| - | + | <br></br> | |
<p><h4>Protocol</h4></p> | <p><h4>Protocol</h4></p> | ||
<ol> | <ol> | ||
Latest revision as of 04:13, 29 September 2011
Notebook
Post-Regionals
Journal
- Defining Objectives
- Techniques Workshop
- Bioinformatics & qRT-PCR
- Sensory Element Fishing
- Electrochem - CPR Oxidation
- Electrochem - Reporter Gene
- Pseudomonas Tools
- Microalgae Tools
- NA Viability Assays
- May - July Group Journal
Protocols
Safety
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.
