神马作文网英语翻译MATERIALS AND METHODSGenetic stocks and growth condition
来源：学生作业帮 编辑：神马作文网作业帮 分类：英语作业 时间：2024/11/17 17:16:46
英语翻译
MATERIALS AND METHODS
Genetic stocks and growth conditions
With one exception,all of the genetic stocks described in this paper were in
Columbia background.The T-DNA insertion SALK_035860 was obtained
from the Arabidopsis Biological Resource Center.The insertion
FLAG_173C12 was obtained from the FST project and was characterized
in a Ws genetic background.Plants homozygous for these insertions were
identified by PCR using allele-specific primers.Seeds were grown on
Metromix 200 (Scotts) and left in 4°C cold room for 2 days before transfer
to growth chambers.Plant age was measured from the time seeds were
transferred to the growth chamber.For phenotypic analysis,plants were
grown in 96-well flats under continuous fluorescent light (100
\3E/minute/m2; Sylvania VHO) at 22°C.Abaxial trichomes were scored 2
weeks after planting with a stereomicroscope.Flowering time represents the
appearance of the first open flower.
RNA blots
Total RNA was isolated using Trizol (Invitrogen) from shoot apices of plants
grown under short-day conditions (10 hours light:14 hours dark) at 22°C.
Whole seedlings (including cotyledons) were used for 8- to 14-day-old
seedlings,the cotyledons and leaves 1 and 2 were removed from plants
harvested between 18-21 days after planting (dap),and the cotyledons and
leaves 1-4 were removed from plants harvested between 23-28 dap.The
number of leaves present on the shoot apex was determined by comparison
with a growth curve generated by dissecting shoot apices grown under the
same SD conditions.To measure SPL3 mRNA,20 \3g of total RNA was run
on 1.2% agarose gels,transferred to Hybond N+ nylon membranes
(Amersham Pharmacia),and crosslinked under UV light.Hybridizations
were performed at 68°C in PerfectHyb plus buffer (Sigma).The SPL3 probe
was PCR amplified using the primers 5\2 ACGAGAGAAGGCGGAAAAGCACAA
3\2 and 5\2 CGGGATCCCTAAGTCTCAATGCATTTAT 3\2
from a SPL3 cDNA clone and was labeled with 32P-dCTP using Prime-II
Random Primer Labeling Kit (Stratagene).Blots were hybridized for 8 hours
at 68°C,washed once in 2\4SSC and 0.1% SDS solution for 5 minutes at
room temperature,twice in 0.5\4SSC and 0.1% SDS for 20 minutes at 68°C,
and once in 0.1\4SSC and 0.1% SDS for 20 minutes at 68°C,and were
scanned with a Storm 860 (Molecular Dynamics).To measure miR156,50
\3g total RNA was separated on 8 M urea/15% denaturing polyacrylamidegels and electrically transferred to Hybond N+ nylon membranes.Blots were
hybridized with a miR156-complementary oligonucleotide labeled with 32PATP
(New England Biolabs) at 40°C in ULTRAhyb-oligo hybridization
buffer (Ambion,Austin,TX).Blots were washed twice at 40°C in 2\4SSC
and 0.5% SDS for 30 minutes before scanning.
5\2 and 3\2RACE
Total RNA was isolated from leaf or floral tissue as described above.
MATERIALS AND METHODS
Genetic stocks and growth conditions
With one exception,all of the genetic stocks described in this paper were in
Columbia background.The T-DNA insertion SALK_035860 was obtained
from the Arabidopsis Biological Resource Center.The insertion
FLAG_173C12 was obtained from the FST project and was characterized
in a Ws genetic background.Plants homozygous for these insertions were
identified by PCR using allele-specific primers.Seeds were grown on
Metromix 200 (Scotts) and left in 4°C cold room for 2 days before transfer
to growth chambers.Plant age was measured from the time seeds were
transferred to the growth chamber.For phenotypic analysis,plants were
grown in 96-well flats under continuous fluorescent light (100
\3E/minute/m2; Sylvania VHO) at 22°C.Abaxial trichomes were scored 2
weeks after planting with a stereomicroscope.Flowering time represents the
appearance of the first open flower.
RNA blots
Total RNA was isolated using Trizol (Invitrogen) from shoot apices of plants
grown under short-day conditions (10 hours light:14 hours dark) at 22°C.
Whole seedlings (including cotyledons) were used for 8- to 14-day-old
seedlings,the cotyledons and leaves 1 and 2 were removed from plants
harvested between 18-21 days after planting (dap),and the cotyledons and
leaves 1-4 were removed from plants harvested between 23-28 dap.The
number of leaves present on the shoot apex was determined by comparison
with a growth curve generated by dissecting shoot apices grown under the
same SD conditions.To measure SPL3 mRNA,20 \3g of total RNA was run
on 1.2% agarose gels,transferred to Hybond N+ nylon membranes
(Amersham Pharmacia),and crosslinked under UV light.Hybridizations
were performed at 68°C in PerfectHyb plus buffer (Sigma).The SPL3 probe
was PCR amplified using the primers 5\2 ACGAGAGAAGGCGGAAAAGCACAA
3\2 and 5\2 CGGGATCCCTAAGTCTCAATGCATTTAT 3\2
from a SPL3 cDNA clone and was labeled with 32P-dCTP using Prime-II
Random Primer Labeling Kit (Stratagene).Blots were hybridized for 8 hours
at 68°C,washed once in 2\4SSC and 0.1% SDS solution for 5 minutes at
room temperature,twice in 0.5\4SSC and 0.1% SDS for 20 minutes at 68°C,
and once in 0.1\4SSC and 0.1% SDS for 20 minutes at 68°C,and were
scanned with a Storm 860 (Molecular Dynamics).To measure miR156,50
\3g total RNA was separated on 8 M urea/15% denaturing polyacrylamidegels and electrically transferred to Hybond N+ nylon membranes.Blots were
hybridized with a miR156-complementary oligonucleotide labeled with 32PATP
(New England Biolabs) at 40°C in ULTRAhyb-oligo hybridization
buffer (Ambion,Austin,TX).Blots were washed twice at 40°C in 2\4SSC
and 0.5% SDS for 30 minutes before scanning.
5\2 and 3\2RACE
Total RNA was isolated from leaf or floral tissue as described above.
材料和方法 基因股票和成长情况 有一例外,所有基因股票被描述在本文里是 哥伦比亚背景.T-DNA 插入SALK_035860 被获得了 从Arabidopsis 生物资源中心.插入 FLAG_173C12 被获得了从FST 项目和被描绘了 在Ws 基因背景中.植物homozygous 为这些插入是 由PCR 辨认使用等位基因具体底漆.种子增长 Metromix 200 (Scotts) 并且离开在4.C 冷室2 天在调动之前 对成长分庭.植物年龄被计量从时间种子是 转移到成长分庭.为显形分析,植物是 增长在96-well 舱内甲板在连续的荧光灯之下(100 E/minute/m2; Sylvania VHO) 在22.C .离开轴心的trichomes 被计分了2 几星期在种植以stereomicroscope 以后.开花的时间代表 第一开放花的出现.
核糖核酸污点 总核糖核酸被隔绝了使用Trizol (Invitrogen) 从植物射击尖顶 增长在短天之下适应(10 个小时light:14 小时黑暗) 在22.C .整体幼木(包括盾状体) 被使用了为8 对14 天 幼木、盾状体和叶子1 和2 从植物被去除了 收获在18-21 天在种植以后(dap),和盾状体之间和 叶子1-4 从植物被去除了被收获在23-28 之间dap .叶子的数字当前在射击尖顶由比较确定了 与成长曲线由解剖引起射击尖顶增长在之下 同样SD 情况.测量SPL3mRNA,
20总核糖核酸g 跑了 在1.2% agarose 胶凝体,转移到Hybond N+ 尼龙膜 (Amersham Pharmacia),和交互相联在紫外光之下.杂交 执行了在68.C 在PerfectHyb 加上缓冲(斯格码) .
从SPL3 DNA 克隆和被标记了与32P-dCTP 使用头等II 任意底漆标记的成套工具(Stratagene) .污点杂交了8 个小时 在68.C,污点是 杂交与miR156 补全低聚核苷酸被标记与32PATP (新英格兰Biolabs) 在40.C 在ULTRAhyb-oligo 杂交.
总核糖核酸与叶子或花卉组织如上所述被隔绝了.
核糖核酸污点 总核糖核酸被隔绝了使用Trizol (Invitrogen) 从植物射击尖顶 增长在短天之下适应(10 个小时light:14 小时黑暗) 在22.C .整体幼木(包括盾状体) 被使用了为8 对14 天 幼木、盾状体和叶子1 和2 从植物被去除了 收获在18-21 天在种植以后(dap),和盾状体之间和 叶子1-4 从植物被去除了被收获在23-28 之间dap .叶子的数字当前在射击尖顶由比较确定了 与成长曲线由解剖引起射击尖顶增长在之下 同样SD 情况.测量SPL3mRNA,
20总核糖核酸g 跑了 在1.2% agarose 胶凝体,转移到Hybond N+ 尼龙膜 (Amersham Pharmacia),和交互相联在紫外光之下.杂交 执行了在68.C 在PerfectHyb 加上缓冲(斯格码) .
从SPL3 DNA 克隆和被标记了与32P-dCTP 使用头等II 任意底漆标记的成套工具(Stratagene) .污点杂交了8 个小时 在68.C,污点是 杂交与miR156 补全低聚核苷酸被标记与32PATP (新英格兰Biolabs) 在40.C 在ULTRAhyb-oligo 杂交.
总核糖核酸与叶子或花卉组织如上所述被隔绝了.