Minute™ Chloroplast Isolation Kit

Invent Biotechnologies Minute™ Chloroplast Isolation Kit is composed of optimized chloroplast isolation buffers and filter cartridges with 2.0 ml collection tubes. The kit is designed to rapidly isolate intact chloroplasts from fresh plant tissues (leaves, seeds and soft stems etc.). Due to the use of filter cartridges with pre-defined pore size and thickness, intact chloroplasts can be isolated from 5-200 mg fresh plant tissues in less than 5 min. Unlike many other methods that require 1-10+ gram tissues for chloroplast isolation, this kit can quickly obtain 1 X 106 to 1 X 107 intact chloroplasts (>90% intact) from fresh plant leaves.

Application
Minute™ Chloroplast Isolation Kit is designed to rapidly isolate intact chloroplasts from small samples (5-200 mg) of fresh plant tissues for applications such as biochemical analysis, SDS-PAGE, immunoblottings, enzyme assays etc. Isolated chloroplasts are ideal starting materials for purification of RNA and DNA for molecular biology studies.
Buffer Formulation: Proprietary

Kit components
1. 25 ml buffer A
2. 25 ml buffer B
3. 50 protein extraction filter cartridges
4. 50 collection tubes with cap
5. Plastic rod (4)


Additional Materials Required
Table-Top Microcentrifuge
1 X PBS or 1 X TES buffer

Protocol

1. Pre-chill buffers and the filter cartridge in collection tube on ice.
2. Place 5-100 mg fresh plant tissue in the filter. For plant leaf, fold or roll the leaf and insert it into the filter. Punch the leaf in the filter repeatedly with a pipette tip for 60 times to reduce the volume (for tissues less than 50 mg punching with the tip is not necessary). For seeds and soft stems, cut with a sharp blade into smaller pieces and place in the filter cartridge(s).
3. Add 200 μl cold buffer A to the filter (Important: shake the bottle vigorously for a few seconds prior to pipetting). Grind the tissue with a plastic rod (included) for 50-60 times with twisting force (Note: The plastic rod is reusable.
For cleaning, rinse it thoroughly with distilled water and dry it with paper towel).
4. Cap the filter and centrifuge in a microcentrifuge at 5,000 rpm at 4oC for 1 min.
Remove the supernatant and resuspend the pellet in 500 μl cold buffer B by pipetting up and down or vortexing.
5. Centriduge at 5,000 rpm for 1 min at 4oC. Remove the supernatant and resuspend the pellet (this is isolated chloroplasts) in 100 to 200 μl cold TES or PBS.

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