sucrose gradient for isolating chloroplasts - (Sep/17/2008 )
Hello everyone,
I would like to do isolate chloroplasts for sequencing and have been doing some research on chloroplast isolation using a sucrose gradient. The information that I find is so different from one paper or protocol to the next that I am very confused. Also, most, if not all, of the protocols deal with larger volumes than what I'm using (such as 50 mL tubes when I'm using 1.5 mL centrifuge tubes).
So, a few questions,
1. when resuspending a pellet in a wash solution, does the pellet have to be "dissolved" into the solution. when i pipet my pellets up and down, they break up into a few pieces but never fully disappear into the wash buffer.
2. is it possible to work with such small amounts in 1.5 mL tubes rather than 50 ml tubes? i'm working with small pieces of algae.
3. how does one figure out the optimum centrifugation speed? it seems from what i've read that different machines require different speeds.
4. i've seen so many different recipes for sucrose solutions. is it okay to just make my sucrose solution with my wash buffer? if i remember correctly it contains EDTA, MgCl2, Hepes, BSA. pH 7.6
Sorry for all the questions and thank you for any information that you can provide.
Hi,
The pellet that you wish to separate using gradient centrifugation has to be resuspended in the sucrose buffer. We do it int helowest % sucrose.
Yes, you can use smaller tubes for smaller volumes. i have used smaller tubes for centrifugation (these tubes hold upto 2ml)
You need to figure out the optimum centrifugation speed (x g) for your experiments. We use something like 150,000x g
Also the buffer you need to figure out from literature. We make up sucrose gradients in tris (final conc of tris 50mM).
Good Luck !!!
The pellet that you wish to separate using gradient centrifugation has to be resuspended in the sucrose buffer. We do it int helowest % sucrose.
Yes, you can use smaller tubes for smaller volumes. i have used smaller tubes for centrifugation (these tubes hold upto 2ml)
You need to figure out the optimum centrifugation speed (x g) for your experiments. We use something like 150,000x g
Also the buffer you need to figure out from literature. We make up sucrose gradients in tris (final conc of tris 50mM).
Good Luck !!!
Well, i played around and managed to get it to work for me except that I had stored the chloroplasts in the fridge for two days before running the sucrose gradient. I ran the gradient anyway and got a whole bunch of "dirty" and broken chloroplasts. Some good ones too.
I tried it again today and went straight into the gradient without storing the chloroplasts. I got nice bands. But when I washed the bands and centrifuged them I didn't get any pellets at all. I kept increasing the centrifiguation, both rcf and the time, but never did i get any pellets.
Hmm...back to the drawing board.!