| RLGS | 点击： 作者： 来源： 时间： 2007-04-10 本站论坛
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|  | 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).
B. Preparation of gel holder
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.
C. Preparation of 1-D Gel
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) .
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