Questions in collection and preservation of plant samples - Purpose: DNA Extraction for DNA fingerprint (Jun/14/2008 )
Hi everybody….am a new member
Hope to get benefit from each other and share our knowledge…..
Here are some questions :-
note:- mango leaves are the samples
Q1:- As I read it is prefer to collect juvenile leaves (young ones), some mango cultivars are characterized by the present of anthocyanine pigment in the early developing stages of leaves
So it is the best choice to be collected for DNA extraction,, Right?
Q2:- Some times the stuff who collects the samples cannot find young leaves because of various reasons. So they will just collect leaves from the available stage of development, from your experience in DNA Extraction and PCR, did you find that the type of leaves will generally affect the amount and quality of DNA extracted and therefore affect the results after PCR?
Q3:- Regarding preservation of samples, did you find that long time preservation will affect the quality of DNA?
Q4:-And is it prefer to grind the samples directly and stored in (-60oC to – 80o C)? Or there is no different if the samples are kept as leaves and grinded only before analysis?
Q5:-Is it safe to store the samples (full leaves or powdered) in -20oC for long period?
Answers are 1) depends, 2) depends, 3) depends, 4) depends 5) depends and the reason for all this is... what is the aim of your expt?
In theory, it will not matter when you collect samples from the trees, the DNA shouldn't change, but if you want to later look at RNA expression of anthocyanins, then yes, it will matter. Also...
The type of leaf shouldn't matter either, the DNA is the same, unless for some reason there are a lot of DNA degrading products or things that will interfere with the extraction in the older leaves, but it is always best to be consistent with your sampling procedures. PCR can be problematic, depends on the plant type, genes being studied etc.
Storage depends on the type of storage and the plant, how dry the leaf is, in ethanol, fixed formaldehyde, whatever... it is totally dependent on the storage.
Grinding or not? Depends, how long between grinding and freezing? what will grinding release that might damage the DNA? how much storage space do you have? Labeling? How much is needed for an experiment? Repeated freeze/thaw if you need to re-extract?
I guess you can probably figure out what I would say for the last one too...
How long? will it affect future experiments with a little degradation? BTW, powdered would definitely be a NO for this one.
1. I'd prefer young leaves. Tissue from young leaves are not tough and hardy and DNA yield is higher per tissue mass (e.g. ug DNA per gram tissue) as compared to senescent leaves. But still, depends on what you want to do with the DNA and whether your focus is on young or senescent leaves.
2. Same as above. For young leaves, you could procure it from seedlings if possible. I won't go after young leaves from a full grown tree as it's very dangerous and cumbersome. There are papers that demonstrated isolation of DNA from senescent leaves and also subsequent PCR amplification. So, it's amplifiable.
3. I had kept frozen leaves for more than a year and the quality was still fine. Kept it at -80 degree Celcius after snap freezing it in liquid Nitrogen.
4. I would prefer not to grind it and store. Ground tissue is easily thawed during handling or transfer and might be a source of DNA degradation, unless lysis buffer can be added immediately. The most I'd just crumple it and store.
5. I won't store the leaves at -20 C.
1bob & BioWizard v0.0.1 thank you very much
In brief: the purpose of the project is to fingerprint various types of mango cultivars using microsatellites markers in order to compare b/w them and to study the Phylogenetic relationships.
We are still in the stages of sample collection, storage and DNA extraction.
What I mean by preservation is freezing the samples (fresh leaves) after collecting them from the field until analysis.
(I won't store the leaves at -20 C.)
Is storage at this temperature will adversely affect the samples?? because we have a problem in -80C freezer and we have store the samples in -20 C until fixing the problem!!!!
Q : during transportation from the field until the lab, the leaves are preserved in bags and saved in cool box,
I know that once the leaves are removed from the tree the enzymes will start degrading the plant cells and damaging DNA.
What is the condition of the leaf that insures that the leaf is still proper for DNA extraction?? (Before and after freezing)
I've transported samples (leaves, seeds etc) from field to lab within 3 hrs without having any adverse effect on DNA quality of isolates. I kept it at cool box. If possible, get a container for liquid nitrogen and a small plastic bags. Seal the clean leaves in the bags, label and place it into the liquid nitrogen container. I preferred the liquid nitrogen container for carrying my samples but cool box works too.
BioWizard v0.0.1 Thank you