We taught students of DNA extraction from leaves of rice seedlings. The first class got pretty good yield, with a lot of DNA precipitates clearly visible after adding ethanol 75%. Two weeks later, we used the same plants and protocol to teach another 2 classes. This time, both classes got very low yield, no visible DNA precipitates, even though we made the students grind the leaves pretty carefully. Everything we did was the same, even the chemicals were taken from the same stocks (which were checked before used).
What could be the reason for the sudden drop in DNA yield? The only differences we could think of were the volumes of SDS lysis buffer (first class we used 600 µL but later classes used 800 µL) and the ages of the seedlings (we used the very same seedlings, so they were 2 week old for first class but 4 week old for later classes, although we did tell the later classes to take the younger leaves).
The basic protocol:
- Grind 3−4 leaves in 600-800 µL lysis buffer containing SDS, Tris HCl (pH 8), EDTA, NaCl.
- Incubate 60 °C, 5 min.
- Centrifuge. Withdraw supernatant into 400 µL phenol:chloroform:isoamyl alcohol (25:24:1). Mix softly.
- Centrifuge. Withdraw top layer into 400 µL isopropanol. Incubate −20 °C, 15 min.
- Centrifuge. Add 1 mL ethanol 75% into precipitate. Mix softly.
- Centrifuge. Dry the precipitate and add ~ 20 µL water to dissolve.
There could be several reasons for the sudden drop in DNA yield in the later classes. Here are a few possibilities:
To troubleshoot the issue, here are some steps you can take:
Possibly also the ethanol has been left open and absorbed water from the air so the final precipitation step may be low efficiency. Try using fresh ethanol
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