Post date: Jun 30, 2014 10:47:52 PM
30 June 2014:
Used the ArchivePure DNA Purification (5 prime) kit, protocol for plant tissue (page 61 of manual).
Used the plant that we started growing with Mari in the greenhouse in January, then it grew mostly in Zach's office.
Samples:
AD1 = took leaves from a sample that was dried on 21vi14, taken from sprig marked with yellow tape, 0.01g of dried tissue
For the following three samples, I first wiped leaves down with ethanol to remove pests (aphids, millibugs?, cobwebs), then removed 4-6 fresh leaves from sprig marked with twist tie:
AF1 = 0.0284g of fresh tissue
AF2 = 0.0279g of fresh tissue
AF3 = 0.0260g of fresh tissue
After adding the DNA Hydration Solution, I kept out the sample at room temperature for rehydration. The next morning, I could still see some of the pellet, so I spun them down at 15,000 x g for 5 min and kept the supernatant. Peter will run a gel in the next few days.
Alfalfa was then transplanted to our garden at home.
If we have to redo this protocol, here are a few thoughts:
-I'd use the low end of the range of the amount of tissue they call for (i.e., 10 mg fresh, 5 mg dried).
-I'd homogenize more than they call for.
-I'd centrifuge at 16,000 rcf.
-I'd let the ethanol evaporate off until you can't really see any left (which will take much longer than 5-10 min).