Hi,
I have to perform genomic DNA extraction from mulberry leaf samples that have been stored for a couple of years in ethanol (unknown concentration) presumably at RT so that part of the samples were not covered by ethanol anymore.
Normally what I do is liophylizing such leaves and then performing extraction using a kit or the standard CTAB method, but with those samples I repeatedly failed (agarose gel empty, spectrophotometer notworking so I cannot assess concentration)...
can anyone suggest me some effective way to get some clean DNA out of them?
many thanks for your suggestions
are you grinding the leaves very small before the dna extraction?
If you are doing a small number of pcrs then running a gel uses a lot of dna to see it if it is degraded but it will pcr well so forget the visualisation and run a sample of your dna to see if it amplifies...it does not take much and even degraded dna usually amplifies well
are you grinding the leaves very small before the dna extraction?
If you are doing a small number of pcrs then running a gel uses a lot of dna to see it if it is degraded but it will pcr well so forget the visualisation and run a sample of your dna to see if it amplifies...it does not take much and even degraded dna usually amplifies well
How much leaf sample you are taking for DNA Extraction? Take enough samples for extraction. Just dont take 100mg or 200 mg as given in protocol. Take one are two grams of leaf samples and grind it with morter and pestle by useing liquid nitrogen.
You mentioned that you don't know the ethanol concentration, which is necessary. Ethanol concentration is not the same for the preservation of DNA and tissue sample.
Take more samples and run the protocol. and adjust your gel runtime. If these experiments failed, then you have to manage the spectrophotometer. Hope it works. Otherwise, you may need new samples.
Hi thanks all for your suggestions. Samples have been grinded with TissueLyser and tungsten beads, so should not be the problem; PCR weakly works so maybe are also some contaminants (polyphenols maybe). Loading extracted DNA concentrated on speedvac on a gel I see some faint degraded band, but I would need more DNA for downstream analyses. I was wondering whether EtOH storage longer than 1 year could cause some problems.
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