D:\116104845.doc 15 March 2007
17 May 2005 - Franks Lab
Leaf DNA prep with Edwards buffer
for this extraction you will need:
Edwards buffer
Buffered Sevag [i.e. phenol/chloroform/isoamyl alcohol (25:24:1)]
locking eppendorf tubes
glass beads (2.5 mm)
liquid nitrogen
1. Use the eppendorf tubes with the locking lids to prevent them popping open
2. Put three glass beads (2.5mm) into each tube 3. label each tube and label plants
4. put several young leaves from each plant into the first tube (older leaves work but
are harder to break up) using forceps to pick leaves.
5. immediately freeze this tube in liquid nitrogen - allow leaf to totally freeze about
1 minute
6. wipe off the forceps with 70% ethanol between new plants - continue collecting
the leaves and freezing the tubes
7. when you have finished collecting leaves from all of the plants, take one tube at a
time and place it in the Silamat S5 shaker and shake for four seconds.
8. Remove the tube and add 400 microliters of Edwards buffer
9. Mix well by vortex or flick with finger as it thaws
10. This tube can sit while you process next tube. (leave at least 3 minutes for
edwards buffer to lyse cells
11. Spin lysed extract in microfuge for 2 minutes full speed (14K)
12. Transfer 350 microliters of lysate to new tube.
13. work in the hood with the phenol, use gloves and a lab coat
14. add 350 microliters of buffered Sevag [phenol/chloroform/isoamyl]
15. snap cap of tube tightly
16. vortex for 10 to 20 seconds each tube. keep you finger on the top of the tube to
minimize the leakage of the phenol.
17. allow tubes to sit for about 5 minutes
18. spin in microfuge for 2 minutes full speed (14K)
19. remove 300 microliters of upper layer to a new tube
20. add 300 microliters of isopropanol alcohol and mix.
D:\116104845.doc 15 March 2007
21. Sit at room temperature for 2 minutes
22. spin in microfuge for 5 minutes full speed (14K)
23. wash two times in 70% ethanol
24. rinse once in 100% ethanol
25. spin final wash and remove last bit of ethanol with pipet man.
26. allow to air dry for 3 to 5 minutes till pellet is whitish
27. resuspend pellet in 50 to 100 microliters of ddH2O.
28. use a pipet tip to resuspend.
29. store the DNA leaf preps in the -20 freezer.
30. pour the phenol phase waste out of each tube and into the liquid organic waste
container in the hood. mark the approximate amount of waste generated on the
sheet on the wall next to the hood. Put the empty tubes (with phenol residue) into
the dry organic waste container.
31. For PCR amplification try using 1 or 2 microlitters of leaf prep for a 20 microliter
reaction
EDWARDS BUFFER
final concentration
reagent
amount to make 100 ml of
solution
200mM
Tris-HCl pH 7.5
20 ml of 1M
250 mM
NaCl
5 ml of 5M
25 mM
EDTA
5 ml of 0.5 M
0.5%
SDS
5 ml of 10%
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ddH20
65 ml