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Troubleshooting DNA extractions:
Click on a question or scroll down to see all the questions and answers:How can I choose which protocol to use?
I get very small amounts or no DNA.
I get DNA but it doesn't digest or PCR.
The protocol calls for grinding with liquid Nitrogen, but I don't have any.
I have a lot of RNA. Should I try to eliminate it, and if so, how?
Q: How can I choose which protocol to use?
A: There are a large variety of protocols available (see the Protocols page). Choose one that has been used on your crop or something similar. Check that you have access to all the required ingredients, or select a different protocol. There is no one best protocol for all situations.
Q: I get very small amounts or no DNA.
A: You may need to grind the tissue longer, or use more tissue. Try to use the youngest tissue possible. The DNA may have degraded if the tissue was thawed and refrozen or kept at room temperature, or if it was ground for a long time without being kept cold. Grinding the tissue is possibly the most important step in extracing DNA, and one of the first places you should troubleshoot if you are not getting good results.
Q: I get DNA but it doesn't digest or PCR.
A: The DNA may have lingering alcohol which prohibits digestion and PCR. Wash carefully with 70% alcohol, let the alcohol evaporate completely before resuspending. If the protocol uses a chloroform step, take only the aqueous layer, do not take up any of the chloroform interfaces.
Q: The protocol calls for grinding with liquid Nitrogen, but I don't have any.
A: There are a number of ways to grind tissue. You can freeze it and then grind with a mortar and pestle, or a drill with a plastic pestle. You can lyophilize (dry) the tissue before grinding. If you are using a young, soft herbaceous tissue (tomato, potato, bean, etc.) you may be able to grind it fresh. There are a number of grinding machines available too, though they may be expensive.
Q: I have a lot of RNA. Should I try to eliminate it, and if so, how?
A: There are varying opinions on whether RNA in with your DNA causes an problems with PCR. Most people ignore the RNA, with no problems. However, if you quantify (measure the concentration of) your DNA using absorbance measurements (with a spectrophotometer), the RNA can lead you to overestimate your DNA concentration.
If you do want to get rid of the RNA in your sample, see our general RNase protocol.
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