Other colleagues are extracting good quality DNA of other crops with the same chemical. so all chemicals are working good.
I would start by preparing a new batch of CTAB. The recommended shelf life is 2 weeks. I had a similar situation and realized I received many attempts later that it was the RNAse A. I moved the RNAse step after phenol chloroform isoamyl alcohol extraction and incubated at 37C for 30 minutes, followed by a chlororm isoamyl alcohol extraction. You can also make sure you are grinding into a fine powder before adding heated CTAB. Increase your incubation time if needed, after adding CTAB.
You can also increase your salting out phase. Try leaving at -20C overnight or you can try letting your pellet dissolve overnight. Are you getting a pellet? If so, what is the consistency?
Some plant hosts are more problematic than others. Maybe add 2% of 2-Mercaptoethanol to the buffer can help. But only add the antioxidant compound to the buffer you are going to use that day, not to all the stock.
Good Luck,
1. Was the tissue from the same plant a few months ago? Was the leaf tissue becoming more difficult to isolate gDNA following the plant development (ex. younger leaves vs. older leaves)?
2. Since your colleague can still use the same extraction butter (or CTAB) to isolate good gDNA, you can choose one day to sit with him/her and do an extraction side-by-side. To see whether anything you missed.
i am taking younger leaves as my samples and i am taking sample from the same tree as i was taking before. i already had tried to extract DNA side by side with my colleague but there is no result.
i am using 2% Ctab. can i increase it to 4 or 6% ?
i allways use CTAB at 2%, i remember to use a few ul of b-mercapto etanol, i dont have a protocol now, but i can search it
I do 2 % CTAB.
During the salting out phase, before incubation, do you see bubbles and strings in the tube? After the washing step, do you see a pellet? If so, what color? Are you stopping if you don't see a pellet?
Also, have you tried quantifying your DNA? If so, what do the curves look like?
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