橡ミヤコグサ形質転換法・秋山
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- ひでたつ かたいわ
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1 Page akiyama@biochem.osakafu-u.ac.jp VA Plant J., 2, (1992) Handberg K. and Stougaard J. Exp. Bot., 48, (1997) Stiller J. (Gresshoff PM) ( ) < ecotype > (Lotus japonicus) Gifu B-129 Agrobacterium tumefaciens GV3101 C58C1 Rif r ( ) (pmp90 Gm r, deletion of T-DNA of ptic58) Mol. Gen. Genet., 204, (1986) Agrobacterium tumefaciens LBA4404 (Clonetech ) pbgi, pgi ( ) pbgi pbi121 Clonetech gus p35s GUS INT (Mol. Gen. Genet., 220, (1990) Max-Planck Willmitzer L. ) gus pgi -gus fusion pbgi 35S CaMV Hind III, Xba I Klenow Xba I, Bam HI, Sma I
2 Page 2 (1) ENOD12A (2) RD29A 5 0.7% 5 1/10X B5 ( )+0.5 µg/ml BA µg/ml NAA X B µg/ml BA µg/ml NAA +10 mm NH µg/ml geneticin base +200 µg/ml ( ) 2 +5 µg/ml geneticin base +100 µg/ml µg/ml 3 1X B µg/ml BA +100 µg/ml 2 3 1X B µg/ml 1 1/2X B µg/ml NAA 2 3 1/2X B
3 Page 3 (1) % EtOH-H 2 O 1 2% NaClO % / *1 4. * / *1 ( 80 ) *2 (2) 1. pbgi pgi Agrobacterium tumefaciens GV3101 C58C1 50% 5 µl 50 µg/ml YEP 2 ml 15 mm 13 cm rpm ( 20 ) * OD YEP OD ml *3 (3) cm No (2)-4. 8 ml 3. (1)-5.
4 Page mm 5. No / pbgi 5,6 *4 GUS GUS *4 GUS X/X GUS GUS (2) LBA4404 (4) GUS GUS ( ) (1) rpm 22 YEP/Km 50 5 ml in 100 ml OD ( ) OD660 GV3101 C58C1/p35S GUS 0.3 5/5 4/6 3/6 INT /6 5/6 4/6 (2) LB/Km 50 9cm GV3101 0/6 3/5 C58C1/pBGI LBA4404/pBGI 0/6 0/5 (3) rpm 2 YEP/Km 50 2 mlin 15mmI.D.X13 cm OD (1/10 2.9) OD
5 Page 5 7 GV3101 C58C1/p35S GUS INT 7 1/5 (4) rpm 21 YEP/Km 50 2 mlin 15mmI.D.X13 cm OD (1/10 ) OD GV3101 C58C1/pBGI 0/10 0/10 (5) rpm 22 YEP/Km 50 2 mlin 15mmI.D.X13 cm OD (1/10 ) OD GV3101 C58C1/pBGI 3/6 2/5 *5 (4) mg/l 9 cm 3 9cm
6 Page / *5 YEP YMB ( ) 300 µg/ml 200 µg/ml (5) / (6) * / / *6 geneticin base 5 1
7 Page 7 (7) * / * /22 8 *7 ( ) *8 1 (8) 1. 5 mm * / * /22 8 *9 NAA *10 80%
8 Page 8 (9) * cm ml / *12 5. *11 *12 1 *13 (10) 1. (1 3 ) DNA 2. gus5 (5 CCATCGCAGCGTAATGCTCT3 ), gus6 (5 GCCGACAGCAGCAGTTTCAT3 ) PCR gus * min, 95 30sec 55 30sec 72 1 min *13 geneticin 50% *14 gus Taq polymerase DNA -gus fusion (5 - CTCGTATGTTGTGTGGAATTGT, 3 - TCACGGGTTGGGGTTTCTAC) 95 1min, 95 30sec 55 30sec 72 X min 30-35
9 Page 9 1 L 1. (geneticin base, ) ( Phytagel) Milli-Q 2. ph 0.5N KOH, HCl ( ) Milli-Q IWAKI SH X20 mm ml IWAKI CUL-JAR300 ( ) 100 ml 2 4 (BA, NAA) DMSO 10mg/ml -20 (NH 4 ) 2 SO 4 4 Phytagel( ) Wako 1 ( ) (J. Exp. Bot., 48, (1997)) Sigma A g 32,200 Phytagel Sigma P-8169
10 Page g 18, % 1/5 Wako Agrobacterium Sigma C g 20,400 5 g(0.5 g X10 ) 7,830 (1/10 ) 1. code No. ( ) for 1 L 1/10X B5 Sigma G-5768 (31 g) 0.31 g 1/10X B5 100X 1 ml 5 mm MES DOJINDO (25 g) 1.1 g 0.5 µg/ml BA Wako (1 g) 500 µg 0.05 µg/ml NAA Wako (25 g) 50 µg 0.6% Wako (500 g) 6 g ph 5.2 B5 (100X -20 ) code No.( ) X100 for 100 ml 100 µg/ml Wako (25 g) 1 g 1 µg/ml Wako (25 g) 10 mg 1 µg/ml Wako (25 g) 10 mg 10 µg/mlwako (25 g) 100 mg
11 Page X B5 3.1 g 1X B5 10 ml 2% ICN (Wako ) 20 g 10 mm (NH 4 ) 2 SO 4 Wako (500 g) 10 ml 400 mm(5.36 g/100 ml) 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel Sigma P g 2 g 200 µg/ml Hoechst 200 mg 0.5 gx10 3. geneticin base (Sigma G-1279, 250 mg) 5 µg/ml 1X B5 3.1 g 1X B5 10 ml 2% 20 g 10 mm (NH 4 ) 2 SO 4 10 ml 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel 2 g 200 µg/ml 200 mg 5 µg/ml geneticin base Sigma G-1279 (250 mg) 5 mg
12 Page ( 100 µg/ml ) 1X B5 3.1 g 1X B5 10 ml 2% 20 g 10 mm (NH 4 ) 2 SO 4 10 ml 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel 2 g 100 µg/ml 100 mg 5 µg/ml geneticin base 5 mg 5. ( geneticin base ) 1X B5 3.1 g 1X B5 10 ml 2% 20 g 10 mm (NH 4 ) 2 SO 4 10 ml 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel 2 g 100 µg/ml 100 mg
13 Page X B5 3.1 g 1X B5 10 ml 2% 20 g 0.2 µg/ml BA 200 µg 0.2% Phytagel 2 g 100 µg/ml 100 mg 7. ( BA ) 1X B5 3.1 g 1X B5 10 ml 2% 20 g 0.2% Phytagel 2 g 100 µg/ml 100 mg 8. 1/2X B g 1/2X B5 5 ml 1% 10 g 0.5 µg/ml NAA 500 µg 0.4% Phytagel 4 g
14 Page ( NAA ) 1/2X B g 1/2X B5 5 ml 1% 10 g 0.4% Phytagel 4 g 10. YEP for Agrobacterium Bacto-peptone 10 g 10 g NaCl 5 g 1M MgCl 2 2 ml for 1 L ph 7.2 MLR / W 15 20,000 lux, 150 µe m -2 s -1
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Lotus manual 1/1 (Lotus japonicus) Stougaard Lotus japonicus IMAIZUMI-ANRAKU HARUKO Tel 0298(38)8377 address
Lotus manual 1/1 (Lotus japonicus) 1999 4 1992 Stougaard Lotus japonicus IMAIZUMI-ANRAKU HARUKO 305 2-1-2 Tel 0298(38)8377 e-mail address onko@ss.abr.affrc.go.jp (Lotus japonicus) ( in vitro Mesorhizobium
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Microsoft Word - 表紙資料２－４
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AS LMD Sampling Manual
AS LMD Application Solutions LaserMicrodissection System LMD SamplingManual 2005 1 AS LMD Sampling Manual -------------------------------------------------------------2 --------------------------------------
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Kw 1-12 1-13 Kw van t Hoff 0 0 0 H S d ln K H ln K = + = RT R d(1/ T ) R ln KW 1/T H 0 /R H 0 = 51.0 kj mol -1 HCl H + + Cl - H2O H + + OH - [] [H + ] [OH - ]=10-14 M 2 25 OH - x OH - [OH - ]=x [H + ]
４章 困難な課題への挑戦 核酸結晶学
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水銀分析マニュアル
1960 P197PHg 253.7 nm UV HgP0P 3 1 HgP2+P 253.7 nm 1 1.5 L/ 30 90 1 1 0.1 ng 50 ml 10 (10 cm ) HNOB3B HClOB4B HB2BSOB4B 1 1 5 200 230 30 P 1) 3 1 1 11 a HNOB3B HClOB4B 1 1 100 ml 100 ml HB2BSOB4B HCl 10%
橡H141（成人モニタリング12年まとめ）表紙・目次・
2 12 C NO2SPM C 10 1 V25 NOx 1 10836 NOx 0.767ppm 13860 NOx 0.703ppm (3) 33562 NOx 0.710ppm (4) 33432 NOx 0.800ppm (5) 15392 NOx 0.611ppm (6) 9263 NOx 0.508ppm (7) 8165 NOx 0.284ppm - 1 - (8) 10866 NOx
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2006 Chlamydomonas reinhardtii RNA 1070010 2007 3 20 p.3 p.3 p.4 p.7 p.8 p.8 p.9 p.9 2 [ ] RNA RNA RNA aada RNA RNAi-37 aph DNA Tag RNA Tag RESDA-PCR Tag Chlamydomonas reinhardtii Tag RNAi 176-10G Tag
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