quick dna miniprep for pcr analysis of plant tissue
- flame blue tips and allow to cool, autoclave.
- take some tissue from a single plant using the lid of an eppendorf tube.
- crush the fresh tissue with the flamed blue tip.
- promptly add 200 µl of 2x ctab buffer, vortex, place at 65ºC at least 5 min (after buffer is added samples can be left at the bench for several hours and at 65ºC o/n if necessary).
- add an equal volume of chloroform, vortex vigorously, centrifuge 1-2 min.
- transfer the supernatant to a fresh tube. avoid transferring interphase which contains polysaccharides and ctab.
- add three vol of etoh, place at –20ºC for 20 min.
- centrifuge for 15 min, rinse pellet with 70% etoh.
- remove etoh, air dry for 5 min at rt (better: speed vac).
- resuspend in 50 µl (20-100 µl depending on pellet size) dh2o, use 3 µl per pcr reaction or quantify on gel and adjust concentration.
- it takes about 1 hour to prepare 20 samples up to the chloroform extraction.
most tissues work, apices, freh leaves, and flowers are very good, old yellow leaves work, and siliques are ok but not optimal. 2-3 apices, one leaf disk the size of the eppendorf tube lid are the amount to start with. when starting with more tissue, resuspend in a larger volume. freezing tissue with liquid nitrogen prior to crushing gives good results and larger yields, so it is recommended to resuspend pellets in 100 µl.
- 2% ctab (w/v)
- 100 mM tris (pH 8.0)
- 1.4 M NaCl
- 1% pvp (polyvinyl-polypyrrolidone) mr 40000
can be aliquoted and autoclaved