用叶片直接PCR,超爽!!!!

刚刚看到的文章，喜欢的话多多支持啊，请斑竹加分啊。
LEAF PCR PROTOCOL
(Klimyuk et al., 1993, TPJ 3: 493-494)
1) Samples are harvested in 1.5 ml tubes and stored on ice.
2) 40ul of 0.25N NaOH was added and the samples boiled for 30 sec.
3) 40ul of 0.25N HCl then 20ul Tris mix was added and the samples boiled for another 2 min.
-tissue samples can then be used immediatly or stored at 4 C for several weeks.
-The amount of tissue used in each PCR reaction should not exceed 2mm2 or the reaction will not work. A small amount of treated material can be excised for use in a PCR reaction with a sterile Gilson tip.
PCR reaction conditions are as follows:
total volume= 50ul
for 5.5 reactions
10X buffer 5ul 27.5uls
10mM dNTPs 1.25uls 6.875uls
primer A 2.5uls 13.75uls
primer B 2.5uls 13.75uls
dH2O 38.75uls 213.1uls
taq poly 1.0ul 5.5uls
95 C 10min 1X
95 C 30sec
55 C 30sec
72 C 45sec 30X
72 C 10min 1X
run 15ul on a 2% agarose gel
note: 2.5 times more primer is used and 2 times more taq polymerase in the leaf PCR protocol. If you could get by with less, Jonathan Jones would have done so!
Stocks
0.25N HCl
0.25N NaOH
Tris buffer:
0.5M Tris pH 8.0
0.25% Nonidet P-40
LEAF PCR ON ARABIDOPSIS TISSUE WITHOUT ALKALINE TREATMENT
Preparation of Master mix:
1 x Taq-buffer
1.5 mM MgCl2
200 mM of each dNTP
1 mM each primer
0.5 ml 20 x Taq polymerase
The mix is stored on ice until use.
Preparation of leaf tissue:
Put the leaf in a small Petri-dish. Make a hole in a leaf with the narrow end of the Pasteur pipette (a forceps might be helpful) and place the leaf in a PCR-tube, if necessary by blowing. On ice, add 50 ml the Master mix.
Running the cycles:
Transfer the tubes directly from ice to the prewarmed 94 C block on the Robocycler and run the following cycles:
94 C for 3 min 1 x
94 C for 30 s; Tann.* for 1 min; 72 C for 1 min-1 min 30 s 35 x
72 C for 10 min (optional) 1 x
Appropriate controls:
Positive Negative
For screening transgenics: plasmid ColO
ColO with endogenous primer-set gDNA
gDNA with endogenous primer-set - DNA