This is a protocol that I have used on a number of
different plant tissues that produces a surprisingly
high yield of clean, 'restrictable' DNA which
is entirely suitable for PCR.
I'm sure it would work on peppers, I hope it is useful.
All the best,
Sean.
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Plant leaf mini-prep.
Adapted from an original arabidopsis method obtained
from a Cold Spring Harbour workshop circa 1994.
I have used this on Arabidopsis, antirrhinum, Brassica sp.,
barley, banana, cherry, coffee, maize, and tobacco to
produce reasonably high quantities of good digestable high
m.w. DNA from Arab rosette leaf SIZED tissue samples
from each species.
The only species which has failed for me was Sunflower,
this produced the dirtiest DNA I have ever extracted
(and I have done cockroach!!!)
1. Grind tissue (approx 1-2 sq cm leaf or petal tissue)
in liquid nitrogen with mortar and pestle to a fine powder.
2. transfer powder to eppendorf tube with 750ul extraction buffer (*1)
3. add 50ul 10% SDS, mix, and incubate 70C for 10 min
4. add 250ul 5M K-Acetate, incubate on ice for 20 min
5. centrifuge as for ethanol precipitation (e.g. 10K bench microfuge) 20 min.
6. pipette supernatent, carefully avoiding precipitate (volume
will not be consistent), into a clean eppendorf containing 0.5ml
isopropanol. Incubate at -20C for 30 min.
7. centrifuge as above for 15 mins to produce DNA pellet.
Remove supernatent and air dry the pellet (20-40 mins depending
on your air conditioning...or not).
8. add a suitable volume of T.E. (I suggest 100 ul is normally
convenient) and triturate pellet until dissolved. Leave 10-15
mins to dissolve fully.
9. centrifuge to remove any insoluble rubbish and transfer supernatent
to a fresh tube.
Run 5-10ul of the sample to test for integrity and concentration.
I use 1ul of 100ul for test genomic PCRs and get reproducible results,
although this will depend on the relative genomic complexity of the
species, it appears adequate for everything I've tried.
Sometimes the solution is coloured (yellowish-green), I guess that this is
contaminated with various things, some of which appear to inhibit PCR.
I know that porphyrin rings inhibit PCR as do some phenolics,
however, whatever it is, it can usually be eliminated by a second
precipitation:
9a. add 1/10th volume 3M Na-Acetate and an equal volume of isopropanol.
The DNA can easily be seen coming out of solution, and will centrifuge
down in a 5min spin.
Dry the pellet and redissolve in (e.g.) 100ul T.E.
-------------------------------------------------------------------
*1 Extraction Buffer
100mM Tris pH 8, 50mM EDTA pH 8, 0.5M NaCl, 10 mM betamercaptoethanol.
-------------------------------------------------------------------
Sean May, Warwick, 1995.
--
-------------------------------------------------
Sean May [lsrei at csv.warwick.ac.uk]
Warwick University, Coventry, U.K.
HTTP://www.warwick.ac.uk/~lsrei/index.html
