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 Aand 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).
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.
Rinse the precipitates with 99.5% ethanol.
Dry up the precipitates.
Dissolve the precipitate in 300 ul dH2O (or 1/10 x TE) containing 0.5 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 750 ng/ul for RLGS analysis in this plant.
Ascertain the size of obtained DNA fragments are larger than lamda DNA using 0.5 % agarose gel electrophoresis (100 - 200 kb recommended).
Cut teflon-tubing (NICHIAS AWG-11, inner diameter: 2.4 mm) into 65 cm (cut a top at an angle of about 20 degrees) for gel holder.
insert it into a glass tube from the bottom, and then a sharp tip of the tubing may exert form the top.
Pull out the tip about 2 cm with a plier.
Cut off the top of the tubing leaving about 1 mm in height.
Push the top of the tubing onto a heated tip of a hand welder.
After a few seconds, push the top of the tubing onto a flat and smooth surface.
Cut off the tubing at the bottom to adjust the length of the gel holder to 62 cm.
Connect a 5 ml plastic syringe fitted with a three-way stopcock and the gel holder with 3 cm of silicon tubing.
Suck up the gel solution gradually to reach the height of 61 cm, and tap on the glass by a finger.
Close the stopcock and set it on a double buret clamp on a support stand.
Wait 10 min. to solidify the gel.
Turn the stopcock to make air free.
Remove the stopcock and silicon tubing.
If the gel does not reach the height of 61 cm, put on the gel solution up to the height of 61 cm using a 1 ml syringe with L-needle (19 gauge, right angle cut, 5 cm long) and wait 10 min.
Put 1-D buffer containing 20% sucrose on the gel to avoid drying.
Add 350 ml of 1x 1-D buffer to anodal tank (bottom) and fix the gels on the apparatus (Bio-craft) .
Mix the reaction solution mildly but completely with pipetting.
Centrifuge at 6,000 rpm for 1 min.
Incubate at 37oC for 3 hrs.
Mix a 10 ul DNA sample with a 3.2 ul labelling solution gently with pipetting ten times.
Incubate at 27oC for 30 min.
Add 3 ul Stop solution.
Fill the cathodal tank (top) with 300 ml of 1x 1-D buffer.
Wash out the 1-D buffer containing 20% sucrose from the top of the gel.
Apply 10 ul (about 0.15 ug DNA for rice, or about 0.45 ug DNA for egg plant) sample on a 1-D gel.
Run electrophoresis at 130 Volts (120 - 140 Volts) for 40 hrs (until the center of BPB reaches 40 cm and that of XC reaches 19 cm from the top of the gel)(Photo) .
Siliconize one side (the side with a tapered edge) of a glass plate.
Set up the gel casting apparatus (Bio-craft).
Mix a gel solution and degas it by vacuum with a sonicator for 5 min.
Cover the top of the gel with Kim Towel wetted by 1 x TBE containing 1% glycerin to keep wet.
After electrophoresis remove the cathodal top buffer with aspirator and take out each gel holder.
Expel the gel slowly using a shortened yellow tip on a 1 ml syringe filled with 0.05% BPB water 10x high buffer (HB)(Photo)
Cut off the top 15 cm of the gel at 45o .
Soak the gel in 50 ml tube containing 40 ml of 1x HB.
Shake the tube gently and equilibrate the gel for 10 min.
Change the buffer and equilibrate the gel once again for 10 min.
Pour the equilibrated gel into a stainless tray.
Cut off lower 12 cm of the gel at 90o.
Place the gel noodle (34 cm long corresponding with 500 bp to 7 kb) in a plastic tray of 22(W) x 350(L) x 3(D) mm.
Remove any trace of the buffer.
Pour 6 ml of 1x HB containing 1600 units of MboI and 0.01% BSA (enzyme solution).
Seal the tray with Saran Wrap (incubate at 37oC for 2 hrs set up the electrophoresis apparatus (Bio-craft).
Fill the electrode tanks with 1x TBE buffer (3 L for the bottom tank).
Rinse the top of the 2-D gel with 1x TBE and wipe with Kimwipe after in-situ digestion with MboI expel the gel noodle from tubing into a 50 ml tube containing 40 ml of 1x TBE.
Equilibrate for 10 min.
Discard the buffer and pour the gel noodle onto a plastic board.
Transfer the gel onto the top of 2-D gel.
Connect agarose noodle gel and 2-D gel with 3 ml of connection agarose gel solution using a 5 ml syringe with 20 G needle (Photo).
Overlay dye agarose gel solution using a 5 ml syringe with 20 G needle.
Fill the electrorode tank with 1x TBE buffer (1.5 L for the upper tank).
Run electrophoresis at 135 Volts (130 - 140 Volts) for 40 hrs until the XC reaches about 33 cm from the top (BPB indicates about 65 bp and XC indicates about 260 bp).
Cover the gel with a piece of filter paper (3 MM, 345 mm x 425 mm).
Cut off the gel outside of the filter paper (Photo).
Take up the gel with the filter paper, and lay it down on two pieces of filter paper (3 MM, 360 mm x 440 mm) with the gel side up.
Overlay Saran Wrap (45 cm wide) onto the gel, and then a piece of filter paper (3 MM, 360 mm x 440 mm) on them.
Cut off an excessive area of the Saran Wrap remaining 5 mm edge.
Set the gel on a gel dryer (Bio-Rad, Model 583).
Dry up the gel at 60oC for 1.2 hr.
Set an intensifying screen (on the bottom), the dried gel, and then a piece of filter paper (3M) on them in a film cassette.
Insert an X-ray film (KODAK X-OMAT AR) between the gel and the intensifying screen in a dark room.
Perform autoradiography at -80 oC for 60-64 hr.
Warm the cassette up to the ambient temperature.
Develop, fix and dry the X-ray film in a dark room.
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