This method may be appllicable for many grass species and some other plants. More simplified emthod for wheat DNA is here.
Collect 2-10 g fresh leaves, cut them into pieces (about 1 cm long) and freeze by liquid nitrogen mill frozen leaf pieces with dry ice using a foods-mill (IWATANI Milser IFM-150D) and collect the flour into a 50 ml tube store leaf flour at -40oC until extraction.
Add 20 ml preheated (to about 70oC by micro-oven) extraction buffer A and 50 ul proteinase K (20 mg/ml), and then stir gently using a spatula.
Incubate at 55oC for 30 min.
Add 20 ml chloroform : isoamylalcohol (24:1) and gently shake for 2 hrs.
Centrifuge at 2,800 rpm for 30 min.
Collect supernatant using a sterile transfer pipet (IWAKI 7801-002, cut off 3 cm from the tip).
Take the precipitate using a sterile transfer pipet (IWAKI 7801-002) (Do not suck! Wind the precipitate around the pipet.) or by centrifuging
Dissolve the precipitate in (2/5 x X) ml TE containing 1 M NaCl and RNase A (1 to 10 ug/ml).
Incubate at 55oC for 1 hr.
Add equal volume of 2-propanol.
Mix gently by swinging the tube slowly.
Rinse the precipitate twice by 5 ml 70% ethanol.
Rinse the precipitate by 5 ml 99% ethanol.
Take the precipitate into a 2 ml microtube by decantation.
Dry the precipitate.
Dissolve the precipitate in 100 x X ul dH2O (or 1/10 x TE) containing 1/10 x X ul RNase-It (Stratagene).
Estimate the DNA concentration (by agarose gel electrophoresis with Etidium bromide or by Hoefer Dyna Quant 200) and make a solution adjusted at 100 ng/ul (50 - 250 ng/ul depending on the purpose) for RLGS analysis.
Ascertain the size of obtained DNA fragments are larger than lamda DNA using 0.5 % agarose gel electrophoresis (100 - 200 kb recommended).
In the case of egg plant, for example
This method may be succesfully appllicable for other plants especially when the above-mentioned method gives a dirtily colored DNA solution because of a large amount of polyphenolic substances in the plant organs.
Collect 1 g (or more) fresh leaves, cut them into pieces (about 1 cm long) and freeze by liquid nitrogen mill frozen leaf pieces with dry ice using a foods-mill (IWATANI Milser IFM-150D) and collect the flour into a 50 ml tube store leaf flour at -40 oC until extraction.
Mix 10 ml of an extraction buffer B, 1 g insoluble PVP and 0.2 g SDS in aother 50 ml tube and Incubate the mixture at 56oC in a water bath.
Put 1 g leaf powder into the mixture and stir gently using a spatula.
Immediately add 50 ul proteinase K (20 mg/ml) and mix.
After 10 min. add 200 ul 2-mercaptethanol.
Incubate the mixture for 1-3 hrs.
Cool down to room temperature.
Add 20 ml 24:1 chloroform : isoamylalcohol and gently shake for 2 hrs.
Centrifuge at 2,400 rpm for 20 min.
Collect the supernatant (about 7.5 ml) using a transfer pipet (IWAKI 7801-002, cut off 3 cm from the tip).
If the supernatant is dirtily colored, repeat the chloroform extraction again.
Add equal volume (7.5 ml) of 2-propanol.
Mix gently but completely,and then you can find white precipitates.
Discard the supernatant by decantation (Do not discard precipitates!).
Rinse the precipitates twice with 70% ethanol.
Transfer the precipitates to a 2 ml micro tube by decantation.
