RLGS protocol

The principle and procedures of RLGS method was first described by Hatada et al. (1991) and its improvement was described by Asakawa (1996).     Basically based on their procedures, we have successfully applied RLGS to gentic analysis in plants (Kawase, 1996; Kawase et al., 1999).

Our current RLGS protocol is described here.

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).