RLGS protocol

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A. Preparation of DNA Solution

In the case of rice, for example

　　　 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). Add 2 ml 10% CTAB solution and mix gently. Add 20 ml 24:1 chloroform : isoamylalcohol and gently shake for 2 hrs. Centrifuge at 2,800 rpm for 30 min.
　 Collect supernatant using a transfer pipet (IWAKI 7801-002, cut off 3 cm from the tip). Add 20 ml PPT buffer. Mix gently by swinging the tube slowly. 　 Keep at room temperature overnight. 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.