Sep 24, 2019
Marchantia chloroplast isolation
- 1University of Cambridge
Protocol Citation: Eftychis Frangedakis 2019. Marchantia chloroplast isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.4x6gxre
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 29, 2019
Last Modified: September 24, 2019
Protocol Integer ID: 25310
Keywords: marchantia chloroplast isolation
Abstract
This protocol is a modifiction of the ISOLATION OF MAIZE CHLOROPLASTS FOR PROTEIN IMPORT STUDIES
protocl from Mark Settles (modified from Ken Cline).
Guidelines
*Pre-cool o/n Buffer GR and pestle and mortar.
Materials
MATERIALS
Percoll™ PlusGe HealthcareCatalog #17-5445-01
MiraclothMerck MilliporeCatalog #475855
GR Buffer (1L)
50 mM Hepes-KOH pH7.5 50 ml 1 M Hepes (23.8g/100ml) [238.3 - Melford]
0.33 M Sorbitol 100ml 3.3 M Sorbitol (300g/500ml) [182.2 - Melford]
1 mM MgCl2 1ml 1M MgCl2
1 mM MnCl2 1ml 1M MnCl2 (19.7g/100ml) [197.91 - Sigma]
2 mM EDTA 4ml 0.5M EDTA pH8
add fresh
5 mM Na-ascorbate 1 g Na-ascorbate [Acros Organic]
1% BSA 10g BSA
Prepare 20 ml 30% Percoll solution by mixing 6ml Percoll, 14ml Buffer GR.
Prepare 10 ml 70% Percoll solution by mixing 7ml Percoll and 3ml Buffer GR.
Add 15ml of 30% Percoil soution in a 50ml Falcon tube. Very carefully underlay 6ml of 70% (denser) Percoll solution using a 5ml Gilson pipette (A in Figure).
Keep the gradient on ice until further use.
Weigh 10g of thallus (grown in 12h light : 12h dark cycle – harvest the tissue 2-3 hours at the start of the light cycle - to reduce starch accumulation) (B in Figure).
Homogenize the tissue using a mortar and pestle into 100ml buffer GR (C and D in Figure).
Filter homogenate through two layers of Miracloth into two 50ml falcon tubes (E and F in Figure)
Centrifuge at 1200 g for 7 min (G in Figure)
Remove supernatant and resuspend the pellet carefully in 2ml of buffer GR.
Transfer the resuspended pellet to the top of the Percoll Gradient using a cut-off pipette tip (H in Figure).
Spin at 7000g for 17 min at 4ºC. USE A SLOW ACCELERATION AND TURN DECELERATION OFF. Broken chloroplasts will reside in the top fraction, intact chloroplasts will reside in the interface of the two Percoll layers (I in Figure).
Carefully transfer intact chloroplasts from the interface, to a new 50 mL Falcon tube.
Wash intact chloroplasts with 25ml of Buffer GR (without BSA). This step removes residual Percoll. Centrifuge at 1500g for 5min at 4oC (J in Figure).
Flash freeze pellet if not used immediatly and store at -80oC