给真好处的奴隶主才是好的奴隶主# Chemistry - 化学
d*g
1 楼
目前申请了美国一大学的Visiting scholar，但单位要求必须拿公务护照才能允许保留
职位办理签证。请问有同志有类似经验吗？公务签证办理J-1后能否再以私人护照再办J
-1？公务护照出国后能以私人护照再延期吗？
谢谢各位！
职位办理签证。请问有同志有类似经验吗？公务签证办理J-1后能否再以私人护照再办J
-1？公务护照出国后能以私人护照再延期吗？
谢谢各位！
H*t
2 楼
有没有人从国内带茶叶过来呀？
不知道是不是也需要报关，会被罚款亚?
不知道是不是也需要报关，会被罚款亚?
S*u
4 楼
森马不知何时站了起来，一边将传讯的魔法纸塞进胸甲里，一边用另一只手捂着嘴，一
连打了好几个哈欠，才自言自语地说：“不行，那个小家伙总是会惹来一堆的麻烦，我
还是亲自去看着他比较好。不过他的运气一向还算不错，不应该有事才对……嗯，还是
去看一眼吧，看过了就可以放心了。另外，不知道他变得帅点了没有。哼，我当初还为
他挨过一箭哪！”
在另一个位面，站在荒凉戈壁上的沃尔德同样收到了讯息，但他只是随手一捏，那
张纸竟然在他的巨手中被捏成飞灰！
“哼！臭小子，等你有了歌顿大人的一半力量，再来命令我吧！”
第三个位面，是阿西瑞斯和塞尔冬共同驻守的位面，他们也同样收到了李察的传讯
。但是两个人的反应却截然相反。
“必须回去！歌顿大人曾经救过我的命。”阿西瑞斯缓缓地说，手中的黑暗教典正
快速翻动着。
“如果你真为歌顿大人着想，就应该守在这里，而不是回去拜见那个毛都没长全的
小家伙！”塞尔冬讥讽道。在他的双手中，两把匕首忽隐忽现，有如两尾游鱼，在十指
间飞速穿行着。一缕缕锐利的杀气不断指向阿西瑞斯各处要害。
“你想背叛歌顿大人？”阿西瑞斯的声音带上了一缕寒意。
“不！我从来不会背叛歌顿大人！但是这并不意味着我要同样向歌顿大人的儿子女
儿效忠！”塞尔冬尖声道。
阿西瑞斯沉默片刻，才说：“既然这样，那我回去。”
塞尔冬愤怒地叫着：“你疯了！回去干什么，就为了向那个小东西表示效忠吗？你
走了，这个位面怎么办？你是想让我一个人搞定对方召来的半神分身吗？那东西怎么看
都和诺兰德的传奇差不多了。难道将来有一天歌顿大人回来了，就看到我们在这个位面
被打得只剩下一个前进基地吗？不，说不定连前进基地都会被打下来！”
“我回去看看，没什么事的话很快就会再回来。你……坚持一下吧。”阿西瑞斯说。
“见鬼了！这里的时间流速是诺兰德的五倍！你稍稍耽搁的话，我怎么坚持得了？
你好好看看，看看下面这些军队！这可是我们好不容易培养起来的，他们也是生命，几
万条生命！”
阿西瑞斯深吸了一口气，说：“浮岛那边的状况，我们又不是不知道。你说的这些都是
借口。所以，这次我必须回去。如果你觉得自己一个人应付不了，那么……撤退吧！”
塞尔冬一窒，连连冷笑起来，说道：“好！撤退！我们打了整整半年的仗，一撤退
就把地盘全都还给敌人了。”
阿西瑞斯不再多说，而是打开了黑暗教典，一道传送门在他面前生成，然后他就直
接跨了进去，丝毫不理会塞尔冬在后面的叫骂。
最后，在原本属于熊彼德家族的位面内，龙法师丽娜正在一个临时营地中休息。营
地布置得很仓促，帐篷都扎得看上去不十分齐整，里面躺满了伤兵，到处都是呻吟声，
血腥气四处弥漫。
当侍卫将李察的讯息送来时，丽娜正拿着把银质小刀，一点点挑着大腿伤口里的腐
肉。在那里，一个泛着蓝色光芒的箭头正从丝丝肌肉里露出来。
她接过魔法信纸，在上面扫了一眼，忽然啊哈一声叫了起来：“小家伙回来了？”
只不过她这么一激动，手上的动作稍稍大了点，银刀一下就将箭头挑了出来，痛得
她龇牙咧嘴。箭锋上有着根根倒刺，非常恶毒。原本丽娜小心翼翼地处理着这枚箭头，
结果激动之下动作稍大，挑出来的箭锋上有小半倒刺上都挂着肉丝。
看着那些被拉出来的肉丝，龙法师痛恨得咬牙切齿。她叫来了一名牧师，将受伤的
大腿往他面前一横。
这名牧师已经有一把年纪了，满脸皱纹上全是岁月的刻痕，但是等级却只有八级。
看来直到生命走到尽头的时候，也没有希望越过十级的关口。看到眼前这条修长笔直、
雪白丰腴的大腿，老牧师的喉节上下剧烈滚动了一下，同时身体微弓，以掩饰小腹上某
些明显的变化。那块地方的变化太过剧烈，就连宽大的神袍都遮挡不住。
“治疗术！一次！快点！”丽娜叱喝着。
老牧师呆滞的目光这才恢复正常，连忙微闭双眼，开始念颂咒语，但对他来说应该
很容易的治疗术咒语却接连念错了两次，平空浪费了不少神力。老牧师忽然感到迎面吹
来的风中多了些刺骨的凉意，那是龙法师的杀气。他陡然出了一身冷汗，终于能够专心
把一个治疗术完成。
神术的光芒闪过，龙法师大腿上的伤口处飘起一团淡青色的水雾，皮肉的颜色有些
恢复正常。但明显还是不够。老牧师正想再准备第二个治疗术，却被丽娜打断了：“把
你的神力留着，去治疗那边的重伤员。”
“但是，丽娜大人，您的伤口还需要至少两次治疗术……”
丽娜怒道：“让你去你就去！”
老牧师张皇逃离后，龙法师叹了口气，拿出一瓶治疗药剂，浇在大腿的伤口上。
带着淡淡乳白色的药剂一触到伤口，即刻沸腾，泛起大片泡沫，更不断涌出刺鼻的
味道。丽娜知道，伤口沾染的毒素正在被清除，但是整个过程却痛得她脸都有些白了。
无论是衍生自神术还是炼金术的治疗药剂，其效果均不如牧师直接施放的治疗术，
还往往会引起痛苦。效果越强，痛苦越大。
但是哪怕不是在新位面的开拓期，即便进入了平稳的扩张时期，异位面征战部队能
招募到的牧师还是极为稀少，那个无能好色的老牧师已经是等级最高的一个了。少量的
治疗术，都被龙法师分配给了重伤的战士，如果不是治疗药剂无法遏制中毒的伤口，她
连这次治疗术都不会要。
处理好腿上的伤口，丽娜站了起来，有些忧郁地看了看远方隐隐约约的城市。那里
正盘踞着数量众多的敌人，依托三座魔法塔进行防御，让她也有些发愁。
连打了好几个哈欠，才自言自语地说：“不行，那个小家伙总是会惹来一堆的麻烦，我
还是亲自去看着他比较好。不过他的运气一向还算不错，不应该有事才对……嗯，还是
去看一眼吧，看过了就可以放心了。另外，不知道他变得帅点了没有。哼，我当初还为
他挨过一箭哪！”
在另一个位面，站在荒凉戈壁上的沃尔德同样收到了讯息，但他只是随手一捏，那
张纸竟然在他的巨手中被捏成飞灰！
“哼！臭小子，等你有了歌顿大人的一半力量，再来命令我吧！”
第三个位面，是阿西瑞斯和塞尔冬共同驻守的位面，他们也同样收到了李察的传讯
。但是两个人的反应却截然相反。
“必须回去！歌顿大人曾经救过我的命。”阿西瑞斯缓缓地说，手中的黑暗教典正
快速翻动着。
“如果你真为歌顿大人着想，就应该守在这里，而不是回去拜见那个毛都没长全的
小家伙！”塞尔冬讥讽道。在他的双手中，两把匕首忽隐忽现，有如两尾游鱼，在十指
间飞速穿行着。一缕缕锐利的杀气不断指向阿西瑞斯各处要害。
“你想背叛歌顿大人？”阿西瑞斯的声音带上了一缕寒意。
“不！我从来不会背叛歌顿大人！但是这并不意味着我要同样向歌顿大人的儿子女
儿效忠！”塞尔冬尖声道。
阿西瑞斯沉默片刻，才说：“既然这样，那我回去。”
塞尔冬愤怒地叫着：“你疯了！回去干什么，就为了向那个小东西表示效忠吗？你
走了，这个位面怎么办？你是想让我一个人搞定对方召来的半神分身吗？那东西怎么看
都和诺兰德的传奇差不多了。难道将来有一天歌顿大人回来了，就看到我们在这个位面
被打得只剩下一个前进基地吗？不，说不定连前进基地都会被打下来！”
“我回去看看，没什么事的话很快就会再回来。你……坚持一下吧。”阿西瑞斯说。
“见鬼了！这里的时间流速是诺兰德的五倍！你稍稍耽搁的话，我怎么坚持得了？
你好好看看，看看下面这些军队！这可是我们好不容易培养起来的，他们也是生命，几
万条生命！”
阿西瑞斯深吸了一口气，说：“浮岛那边的状况，我们又不是不知道。你说的这些都是
借口。所以，这次我必须回去。如果你觉得自己一个人应付不了，那么……撤退吧！”
塞尔冬一窒，连连冷笑起来，说道：“好！撤退！我们打了整整半年的仗，一撤退
就把地盘全都还给敌人了。”
阿西瑞斯不再多说，而是打开了黑暗教典，一道传送门在他面前生成，然后他就直
接跨了进去，丝毫不理会塞尔冬在后面的叫骂。
最后，在原本属于熊彼德家族的位面内，龙法师丽娜正在一个临时营地中休息。营
地布置得很仓促，帐篷都扎得看上去不十分齐整，里面躺满了伤兵，到处都是呻吟声，
血腥气四处弥漫。
当侍卫将李察的讯息送来时，丽娜正拿着把银质小刀，一点点挑着大腿伤口里的腐
肉。在那里，一个泛着蓝色光芒的箭头正从丝丝肌肉里露出来。
她接过魔法信纸，在上面扫了一眼，忽然啊哈一声叫了起来：“小家伙回来了？”
只不过她这么一激动，手上的动作稍稍大了点，银刀一下就将箭头挑了出来，痛得
她龇牙咧嘴。箭锋上有着根根倒刺，非常恶毒。原本丽娜小心翼翼地处理着这枚箭头，
结果激动之下动作稍大，挑出来的箭锋上有小半倒刺上都挂着肉丝。
看着那些被拉出来的肉丝，龙法师痛恨得咬牙切齿。她叫来了一名牧师，将受伤的
大腿往他面前一横。
这名牧师已经有一把年纪了，满脸皱纹上全是岁月的刻痕，但是等级却只有八级。
看来直到生命走到尽头的时候，也没有希望越过十级的关口。看到眼前这条修长笔直、
雪白丰腴的大腿，老牧师的喉节上下剧烈滚动了一下，同时身体微弓，以掩饰小腹上某
些明显的变化。那块地方的变化太过剧烈，就连宽大的神袍都遮挡不住。
“治疗术！一次！快点！”丽娜叱喝着。
老牧师呆滞的目光这才恢复正常，连忙微闭双眼，开始念颂咒语，但对他来说应该
很容易的治疗术咒语却接连念错了两次，平空浪费了不少神力。老牧师忽然感到迎面吹
来的风中多了些刺骨的凉意，那是龙法师的杀气。他陡然出了一身冷汗，终于能够专心
把一个治疗术完成。
神术的光芒闪过，龙法师大腿上的伤口处飘起一团淡青色的水雾，皮肉的颜色有些
恢复正常。但明显还是不够。老牧师正想再准备第二个治疗术，却被丽娜打断了：“把
你的神力留着，去治疗那边的重伤员。”
“但是，丽娜大人，您的伤口还需要至少两次治疗术……”
丽娜怒道：“让你去你就去！”
老牧师张皇逃离后，龙法师叹了口气，拿出一瓶治疗药剂，浇在大腿的伤口上。
带着淡淡乳白色的药剂一触到伤口，即刻沸腾，泛起大片泡沫，更不断涌出刺鼻的
味道。丽娜知道，伤口沾染的毒素正在被清除，但是整个过程却痛得她脸都有些白了。
无论是衍生自神术还是炼金术的治疗药剂，其效果均不如牧师直接施放的治疗术，
还往往会引起痛苦。效果越强，痛苦越大。
但是哪怕不是在新位面的开拓期，即便进入了平稳的扩张时期，异位面征战部队能
招募到的牧师还是极为稀少，那个无能好色的老牧师已经是等级最高的一个了。少量的
治疗术，都被龙法师分配给了重伤的战士，如果不是治疗药剂无法遏制中毒的伤口，她
连这次治疗术都不会要。
处理好腿上的伤口，丽娜站了起来，有些忧郁地看了看远方隐隐约约的城市。那里
正盘踞着数量众多的敌人，依托三座魔法塔进行防御，让她也有些发愁。
h*n
5 楼
4种老鼠, 分别带某个基因(KO/KI/tg), 要把四个基因弄到一个老鼠上, 好像逐个杂交
的工作量太大, 而且到最后比例太低. 不知道可行不可行.
各位有没有做过的, 给点指导?
谢谢
的工作量太大, 而且到最后比例太低. 不知道可行不可行.
各位有没有做过的, 给点指导?
谢谢
e*2
6 楼
不然就不是好人
t*i
7 楼
这个，我们单位同事每次回国都带很多。
【在 H********t 的大作中提到】
: 有没有人从国内带茶叶过来呀？
: 不知道是不是也需要报关，会被罚款亚?
【在 H********t 的大作中提到】
: 有没有人从国内带茶叶过来呀？
: 不知道是不是也需要报关，会被罚款亚?
s*r
9 楼
他爸回来不变成明英宗了，谁来做于謙
g*e
10 楼
I did so, with 3 different flox/flox and one cre mice
T*s
11 楼
这里的时间流速是诺兰德的5倍，这对位面征战不是很好么
法罗是诺兰德的10倍，李察还是靠顶级献祭才得到的
法罗是诺兰德的10倍，李察还是靠顶级献祭才得到的
h*n
12 楼
那你到了最后的选老鼠岂不是比例很低? 是不是要很多老鼠来选?
我的有些类似, 一个oncogene, 两个Flox/Flox, 一个Cre老鼠. 其中两个flox/flox我
都要homozygous.
谢谢.
【在 g****e 的大作中提到】
: I did so, with 3 different flox/flox and one cre mice
我的有些类似, 一个oncogene, 两个Flox/Flox, 一个Cre老鼠. 其中两个flox/flox我
都要homozygous.
谢谢.
【在 g****e 的大作中提到】
: I did so, with 3 different flox/flox and one cre mice
T*s
13 楼
回来的三个都是聪明人
李察迷失在乱流中都能回来
还未来的圣构装师
正是拉拢关系的好时候
反正他们还是追随歌顿，歌顿走时也交代了李察回来的话
【在 s*r 的大作中提到】
: 他爸回来不变成明英宗了，谁来做于謙
李察迷失在乱流中都能回来
还未来的圣构装师
正是拉拢关系的好时候
反正他们还是追随歌顿，歌顿走时也交代了李察回来的话
【在 s*r 的大作中提到】
: 他爸回来不变成明英宗了，谁来做于謙
g*e
14 楼
I need homozygous for 2 flox/flox, heterzygous for one flox/flox and cre, it
took me one whole year to get it. The ratio at beginning is very low, but
you can increase efficiency after a few rounds of mating.
took me one whole year to get it. The ratio at beginning is very low, but
you can increase efficiency after a few rounds of mating.
c*r
15 楼
森马回来属于合情合理，她守的位面局势稳定
黑暗牧师是个明白人，他想清楚了利害关系，选择回来
塞尔东，我觉得这个人没有什么远见
沃尔德的性格跟那个刚德有点像，属于一根筋，忠于职守，所以不回来
丽娜还不知道，如果回来的话，多半是因为私人感情。
【在 T*********s 的大作中提到】
: 回来的三个都是聪明人
: 李察迷失在乱流中都能回来
: 还未来的圣构装师
: 正是拉拢关系的好时候
: 反正他们还是追随歌顿，歌顿走时也交代了李察回来的话
黑暗牧师是个明白人，他想清楚了利害关系，选择回来
塞尔东，我觉得这个人没有什么远见
沃尔德的性格跟那个刚德有点像，属于一根筋，忠于职守，所以不回来
丽娜还不知道，如果回来的话，多半是因为私人感情。
【在 T*********s 的大作中提到】
: 回来的三个都是聪明人
: 李察迷失在乱流中都能回来
: 还未来的圣构装师
: 正是拉拢关系的好时候
: 反正他们还是追随歌顿，歌顿走时也交代了李察回来的话
O*e
16 楼
You have to make sure they are not on same chromosomes
【在 h********n 的大作中提到】
: 4种老鼠, 分别带某个基因(KO/KI/tg), 要把四个基因弄到一个老鼠上, 好像逐个杂交
: 的工作量太大, 而且到最后比例太低. 不知道可行不可行.
: 各位有没有做过的, 给点指导?
: 谢谢
【在 h********n 的大作中提到】
: 4种老鼠, 分别带某个基因(KO/KI/tg), 要把四个基因弄到一个老鼠上, 好像逐个杂交
: 的工作量太大, 而且到最后比例太低. 不知道可行不可行.
: 各位有没有做过的, 给点指导?
: 谢谢
h*n
17 楼
在同一个染色体上会咋样?
【在 O******e 的大作中提到】
: You have to make sure they are not on same chromosomes
【在 O******e 的大作中提到】
: You have to make sure they are not on same chromosomes
O*e
18 楼
正常分离的话，你拿不到你要的组合啊。重组几率太低。
【在 h********n 的大作中提到】
: 在同一个染色体上会咋样?
【在 h********n 的大作中提到】
: 在同一个染色体上会咋样?
h*g
19 楼
同问！
【在 h********n 的大作中提到】
: 在同一个染色体上会咋样?
【在 h********n 的大作中提到】
: 在同一个染色体上会咋样?
c*l
20 楼
即使在同一条染色体上还可以发生homologous recombination而组合在一起啊。
只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
是不低的。
The centimorgan is equal to a 1% chance that a marker at one genetic locus
on a chromosome will be separated from a marker at a second locus due to
crossing over in a single generation
One cM is about 1 Mb apart.
【在 O******e 的大作中提到】
: 正常分离的话，你拿不到你要的组合啊。重组几率太低。
只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
是不低的。
The centimorgan is equal to a 1% chance that a marker at one genetic locus
on a chromosome will be separated from a marker at a second locus due to
crossing over in a single generation
One cM is about 1 Mb apart.
【在 O******e 的大作中提到】
: 正常分离的话，你拿不到你要的组合啊。重组几率太低。
O*e
21 楼
I wouldn't count on that. lz doesn't really have to get the homozygotes.
【在 c****l 的大作中提到】
: 即使在同一条染色体上还可以发生homologous recombination而组合在一起啊。
: 只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
: 是不低的。
: The centimorgan is equal to a 1% chance that a marker at one genetic locus
: on a chromosome will be separated from a marker at a second locus due to
: crossing over in a single generation
: One cM is about 1 Mb apart.
【在 c****l 的大作中提到】
: 即使在同一条染色体上还可以发生homologous recombination而组合在一起啊。
: 只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
: 是不低的。
: The centimorgan is equal to a 1% chance that a marker at one genetic locus
: on a chromosome will be separated from a marker at a second locus due to
: crossing over in a single generation
: One cM is about 1 Mb apart.
c*l
22 楼
If you do not count on science, anything else you will count on?
【在 O******e 的大作中提到】
: I wouldn't count on that. lz doesn't really have to get the homozygotes.
【在 O******e 的大作中提到】
: I wouldn't count on that. lz doesn't really have to get the homozygotes.
O*e
23 楼
You missed my point. I wouldn't count on HR for that end. Just too much work
to get what lz wanted.
【在 c****l 的大作中提到】
: If you do not count on science, anything else you will count on?
to get what lz wanted.
【在 c****l 的大作中提到】
: If you do not count on science, anything else you will count on?
g*0
24 楼
a. make sure the four genes are on different chromosome
b. plan the breeding carefully so that for each round of breeding the
mendelian ratio of getting your target genotype is no less than 1:4 ( below
1:16 is almost mission impossible). This way, in general, it takes one round
of breeding to introduce one mutated allele to the genome. Say you are
planning to introduce 4 mutated genes, 2 homozygous 2 heterozygous, you will
need 4 + 2 = 6 round of breeding. Each round will take at least 9 weeks (
b. plan the breeding carefully so that for each round of breeding the
mendelian ratio of getting your target genotype is no less than 1:4 ( below
1:16 is almost mission impossible). This way, in general, it takes one round
of breeding to introduce one mutated allele to the genome. Say you are
planning to introduce 4 mutated genes, 2 homozygous 2 heterozygous, you will
need 4 + 2 = 6 round of breeding. Each round will take at least 9 weeks (
h*n
25 楼
thank you so much for the advice.
As to the time, I am thinking of breeding A+B and C+D at the same time and
then do ABCD, so as to save some time. Do you think there is any potential
issues there?
below
round
will
3
may
very
【在 g*******0 的大作中提到】
: a. make sure the four genes are on different chromosome
: b. plan the breeding carefully so that for each round of breeding the
: mendelian ratio of getting your target genotype is no less than 1:4 ( below
: 1:16 is almost mission impossible). This way, in general, it takes one round
: of breeding to introduce one mutated allele to the genome. Say you are
: planning to introduce 4 mutated genes, 2 homozygous 2 heterozygous, you will
: need 4 + 2 = 6 round of breeding. Each round will take at least 9 weeks (
As to the time, I am thinking of breeding A+B and C+D at the same time and
then do ABCD, so as to save some time. Do you think there is any potential
issues there?
below
round
will
3
may
very
【在 g*******0 的大作中提到】
: a. make sure the four genes are on different chromosome
: b. plan the breeding carefully so that for each round of breeding the
: mendelian ratio of getting your target genotype is no less than 1:4 ( below
: 1:16 is almost mission impossible). This way, in general, it takes one round
: of breeding to introduce one mutated allele to the genome. Say you are
: planning to introduce 4 mutated genes, 2 homozygous 2 heterozygous, you will
: need 4 + 2 = 6 round of breeding. Each round will take at least 9 weeks (
n*k
26 楼
I was gonna reply to you but it is not an easy task, and got a bit busy
today...I would seriously suggest you
take a day or two to sit down:
1: Know your goals(what genotypes do you ultimate need, what's proper
control...do you want Cre
control or not, all will dramatically change your breeding stategies)
2: What mice do you have to start with
3: Money versa time (man power as well---sometimes one more generation
seems longer and wasteful
but indeed could save you tons if you don't h
【在 h********n 的大作中提到】
: thank you so much for the advice.
: As to the time, I am thinking of breeding A+B and C+D at the same time and
: then do ABCD, so as to save some time. Do you think there is any potential
: issues there?
:
: below
: round
: will
: 3
: may
today...I would seriously suggest you
take a day or two to sit down:
1: Know your goals(what genotypes do you ultimate need, what's proper
control...do you want Cre
control or not, all will dramatically change your breeding stategies)
2: What mice do you have to start with
3: Money versa time (man power as well---sometimes one more generation
seems longer and wasteful
but indeed could save you tons if you don't h
【在 h********n 的大作中提到】
: thank you so much for the advice.
: As to the time, I am thinking of breeding A+B and C+D at the same time and
: then do ABCD, so as to save some time. Do you think there is any potential
: issues there?
:
: below
: round
: will
: 3
: may
n*k
27 楼
Just saw u have transgene, in that case, likely it is a lot LOT easier....
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
h*n
28 楼
太感谢了.
我的座位后面有个白板, 我两个月前把breeding strategy写在上面, 然后每天都看看
想想. 主要的问题是, 周围的人都不太有经验, 我有一写经验, 还差很远.
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
我的座位后面有个白板, 我两个月前把breeding strategy写在上面, 然后每天都看看
想想. 主要的问题是, 周围的人都不太有经验, 我有一写经验, 还差很远.
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
h*n
29 楼
Cre control 是指老鼠跟非Cre mice交配作为control吗? 如果是这个, 我不需要.
我要做的最终的比较组是 gene +/+, +/-, -/-即wt, hetero KO, homo KO. 其中KO是
cre干的.
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
我要做的最终的比较组是 gene +/+, +/-, -/-即wt, hetero KO, homo KO. 其中KO是
cre干的.
【在 n********k 的大作中提到】
: I was gonna reply to you but it is not an easy task, and got a bit busy
: today...I would seriously suggest you
: take a day or two to sit down:
: 1: Know your goals(what genotypes do you ultimate need, what's proper
: control...do you want Cre
: control or not, all will dramatically change your breeding stategies)
: 2: What mice do you have to start with
: 3: Money versa time (man power as well---sometimes one more generation
: seems longer and wasteful
: but indeed could save you tons if you don't h
n*k
30 楼
In general, it would be faster if you don't control for Cre...but I
personally don't recommend as Cre could be a
complicating factor...therefore for my final breeding, I always use one with
double Cre while the other one
with no Cre, and then every pups have cre but different genotype, rather
than the other way: every pups is the
same genotype but either with or without cre...in your case, u might want to
keep only one allele different
between experimental groups while keep the rest the same,
eg
【在 h********n 的大作中提到】
: Cre control 是指老鼠跟非Cre mice交配作为control吗? 如果是这个, 我不需要.
: 我要做的最终的比较组是 gene +/+, +/-, -/-即wt, hetero KO, homo KO. 其中KO是
: cre干的.
personally don't recommend as Cre could be a
complicating factor...therefore for my final breeding, I always use one with
double Cre while the other one
with no Cre, and then every pups have cre but different genotype, rather
than the other way: every pups is the
same genotype but either with or without cre...in your case, u might want to
keep only one allele different
between experimental groups while keep the rest the same,
eg
【在 h********n 的大作中提到】
: Cre control 是指老鼠跟非Cre mice交配作为control吗? 如果是这个, 我不需要.
: 我要做的最终的比较组是 gene +/+, +/-, -/-即wt, hetero KO, homo KO. 其中KO是
: cre干的.
h*n
31 楼
I got your points, which are very helpful to me.
I am going back to lab and think of these carefully.
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
I am going back to lab and think of these carefully.
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
h*n
32 楼
I see your points, which are very helpful to me.
I am going back to lab and think of these carefully.
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
I am going back to lab and think of these carefully.
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
D*a
33 楼
就有一个问题，怎么测哪个是cre homozygote啊？定量PCR？
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
with
to
【在 n********k 的大作中提到】
: In general, it would be faster if you don't control for Cre...but I
: personally don't recommend as Cre could be a
: complicating factor...therefore for my final breeding, I always use one with
: double Cre while the other one
: with no Cre, and then every pups have cre but different genotype, rather
: than the other way: every pups is the
: same genotype but either with or without cre...in your case, u might want to
: keep only one allele different
: between experimental groups while keep the rest the same,
: eg
h*n
34 楼
就普通PCR吧. 设计primer跨过endogenous/exogenous sequence, homo/hetero/wt的产
物不同.
【在 D*a 的大作中提到】
: 就有一个问题，怎么测哪个是cre homozygote啊？定量PCR？
:
: with
: to
物不同.
【在 D*a 的大作中提到】
: 就有一个问题，怎么测哪个是cre homozygote啊？定量PCR？
:
: with
: to
D*a
35 楼
你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
另外cre很长的吧？
【在 h********n 的大作中提到】
: 就普通PCR吧. 设计primer跨过endogenous/exogenous sequence, homo/hetero/wt的产
: 物不同.
另外cre很长的吧？
【在 h********n 的大作中提到】
: 就普通PCR吧. 设计primer跨过endogenous/exogenous sequence, homo/hetero/wt的产
: 物不同.
k*o
36 楼
能加段tag吗？
【在 D*a 的大作中提到】
: 你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
: 另外cre很长的吧？
D*a
37 楼
cre不是我们自己做的啊
我倒是知道有人鉴定cre homozygote是靠交配来鉴定的。
按这种思路，如果不牵扯background，倒是可以纯合杂和的cre一起用来交配下一代，
至少可以把下一代的cre+ 提高到 > 50%
【在 k****o 的大作中提到】
:
: 能加段tag吗？
我倒是知道有人鉴定cre homozygote是靠交配来鉴定的。
按这种思路，如果不牵扯background，倒是可以纯合杂和的cre一起用来交配下一代，
至少可以把下一代的cre+ 提高到 > 50%
【在 k****o 的大作中提到】
:
: 能加段tag吗？
h*n
38 楼
如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
【在 D*a 的大作中提到】
: 你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
: 另外cre很长的吧？
【在 D*a 的大作中提到】
: 你的cre是定点插入的？我都不知道我的cre插在哪儿-_-
: 另外cre很长的吧？
D*a
39 楼
不知道cre插在哪儿，怎么看cre+/+ 和cre+/0？？？
【在 h********n 的大作中提到】
: 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
【在 h********n 的大作中提到】
: 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
D*a
40 楼
另外刚跟老板讨论这个问题，如果用cre /cre，cre倒是有control了，可是loxp位点没
有control了，也许你加进去的loxp位点somehow影响基因表达呢？所以我们组用的wt
mice实际上是flox/flox, 作图的时候写"control"，不写"wt"
也有人为了这个会多加一个纯粹的wt/wt作为control group, 用来跟flox/flox比较证
明flox/flox是wt的表型。
【在 h********n 的大作中提到】
: 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
有control了，也许你加进去的loxp位点somehow影响基因表达呢？所以我们组用的wt
mice实际上是flox/flox, 作图的时候写"control"，不写"wt"
也有人为了这个会多加一个纯粹的wt/wt作为control group, 用来跟flox/flox比较证
明flox/flox是wt的表型。
【在 h********n 的大作中提到】
: 如果觉得不保险, 就设计两对primer, 分别看Wt和Cre.
D*a
41 楼
你说的两边分别配ab和cd，到最后一起配abcd是完全可以的
另外你永远也不知道cre插在什么地方，只有通过交配才知道，这个是一个风险
还有就是你现阶段不要只想breeding，可以想一下有什么实验是需要set up但是又不需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
节约老鼠且不说了，反正老板花钱^_^，节约时间是很重要的, 我们一个博士的project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
。
另外你永远也不知道cre插在什么地方，只有通过交配才知道，这个是一个风险
还有就是你现阶段不要只想breeding，可以想一下有什么实验是需要set up但是又不需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
节约老鼠且不说了，反正老板花钱^_^，节约时间是很重要的, 我们一个博士的project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
。
h*n
42 楼
great point.
Thanks
需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练
手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的
验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR
genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，
behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还
有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
【在 D*a 的大作中提到】
: 你说的两边分别配ab和cd，到最后一起配abcd是完全可以的
: 另外你永远也不知道cre插在什么地方，只有通过交配才知道，这个是一个风险
: 还有就是你现阶段不要只想breeding，可以想一下有什么实验是需要set up但是又不需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
: 节约老鼠且不说了，反正老板花钱^_^，节约时间是很重要的, 我们一个博士的project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
: 。
Thanks
需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练
手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的
验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR
genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，
behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还
有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
【在 D*a 的大作中提到】
: 你说的两边分别配ab和cd，到最后一起配abcd是完全可以的
: 另外你永远也不知道cre插在什么地方，只有通过交配才知道，这个是一个风险
: 还有就是你现阶段不要只想breeding，可以想一下有什么实验是需要set up但是又不需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
: 节约老鼠且不说了，反正老板花钱^_^，节约时间是很重要的, 我们一个博士的project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
: 。
g*0
43 楼
You always have to do parallel breeding to save time. But
to get A/A B/+ or C/C D/+ you need two round breeding:
(1)A/+ x B/+ --> A/+ B/+ (1:4) and A/+ x A/+ --> A/A (1:4) (start with one
mutant allele)
(2) A/+ B/+ x A/A --> A/A B/+ (1:4)
if you breed AB with CD:
(3)A/A B/+ x C/C D/+ --> A/+ B/+ C/+ D/+ (1:4) and A/A B/+ x A/A B/+ --> A/
A B/B (1:4)
(4) A/+ B/+ C/+ D/+ x A/A B/B --> A/A B/+/C/+ D/+ or A/A B/B C/+ D/+ (1:8)
(5) A/A B/+ C/+ D/+ x A/A B/B --> A/A B/B C/+ D/+ (1:8) (6 mutated alle
【在 h********n 的大作中提到】
: thank you so much for the advice.
: As to the time, I am thinking of breeding A+B and C+D at the same time and
: then do ABCD, so as to save some time. Do you think there is any potential
: issues there?
:
: below
: round
: will
: 3
: may
to get A/A B/+ or C/C D/+ you need two round breeding:
(1)A/+ x B/+ --> A/+ B/+ (1:4) and A/+ x A/+ --> A/A (1:4) (start with one
mutant allele)
(2) A/+ B/+ x A/A --> A/A B/+ (1:4)
if you breed AB with CD:
(3)A/A B/+ x C/C D/+ --> A/+ B/+ C/+ D/+ (1:4) and A/A B/+ x A/A B/+ --> A/
A B/B (1:4)
(4) A/+ B/+ C/+ D/+ x A/A B/B --> A/A B/+/C/+ D/+ or A/A B/B C/+ D/+ (1:8)
(5) A/A B/+ C/+ D/+ x A/A B/B --> A/A B/B C/+ D/+ (1:8) (6 mutated alle
【在 h********n 的大作中提到】
: thank you so much for the advice.
: As to the time, I am thinking of breeding A+B and C+D at the same time and
: then do ABCD, so as to save some time. Do you think there is any potential
: issues there?
:
: below
: round
: will
: 3
: may
g*0
44 楼
Usually nobody know what locus is the cre located. The safe way to tell cre/
+ or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
enough attention the intensity of PCR genotyping band normally can give you
some clue that two copy PCR band is much brighter than one copy. Better
approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
right cre/+ cre/cre tail DNA control to help you establish the criteria to
judge what is cre/+ or cre/cre (eg. two band wit
【在 h********n 的大作中提到】
: great point.
: Thanks
:
: 需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练
: 手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的
: 验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR
: genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
: project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，
: behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还
: 有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
+ or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
enough attention the intensity of PCR genotyping band normally can give you
some clue that two copy PCR band is much brighter than one copy. Better
approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
right cre/+ cre/cre tail DNA control to help you establish the criteria to
judge what is cre/+ or cre/cre (eg. two band wit
【在 h********n 的大作中提到】
: great point.
: Thanks
:
: 需要最终model的，这些都可以在前期的model上做的，具体比如各种你还不熟的技术练
: 手，abcd蛋白的东西南北blot和IHC的抗体test，某种kit能不能管用，cre的特异性的
: 验证， PCR genotypage的带大小温度，是不是有利于在最终model中combine PCR
: genotypage，省的你一只老鼠三个pcr才知道谁是谁，等等等等，
: project也是要等一年，刚开始觉得等一年太无聊了，现在model两个月之后就出来了，
: behavior的test还毫无头绪，IHC的抗体倒是test了，不过不知道怎么定量分析呢，还
: 有其他乱七八糟的小test。所以你要是能计划好，这些东西可以全部都先做掉
D*a
45 楼
多谢，然后还是应该breeding验证的吧～
cre homo的好处就是maintaining起来比较省事，不用每代都去pcr
cre/
pay
you
the
cre,
different
【在 g*******0 的大作中提到】
: Usually nobody know what locus is the cre located. The safe way to tell cre/
: + or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
: enough attention the intensity of PCR genotyping band normally can give you
: some clue that two copy PCR band is much brighter than one copy. Better
: approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
: right cre/+ cre/cre tail DNA control to help you establish the criteria to
: judge what is cre/+ or cre/cre (eg. two band wit
cre homo的好处就是maintaining起来比较省事，不用每代都去pcr
cre/
pay
you
the
cre,
different
【在 g*******0 的大作中提到】
: Usually nobody know what locus is the cre located. The safe way to tell cre/
: + or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
: enough attention the intensity of PCR genotyping band normally can give you
: some clue that two copy PCR band is much brighter than one copy. Better
: approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
: right cre/+ cre/cre tail DNA control to help you establish the criteria to
: judge what is cre/+ or cre/cre (eg. two band wit
h*n
46 楼
我昨天在想这个Cre的问题。 我想， 能不能用这个transgene头围的序列设计primer测
序， 测出transgene前后的序列， 然后设计primer根据区分homo hetero and wt.
homo和wt都是一条带， hetero两条带。
cre/
pay
you
the
cre,
different
【在 g*******0 的大作中提到】
: Usually nobody know what locus is the cre located. The safe way to tell cre/
: + or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
: enough attention the intensity of PCR genotyping band normally can give you
: some clue that two copy PCR band is much brighter than one copy. Better
: approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
: right cre/+ cre/cre tail DNA control to help you establish the criteria to
: judge what is cre/+ or cre/cre (eg. two band wit
序， 测出transgene前后的序列， 然后设计primer根据区分homo hetero and wt.
homo和wt都是一条带， hetero两条带。
cre/
pay
you
the
cre,
different
【在 g*******0 的大作中提到】
: Usually nobody know what locus is the cre located. The safe way to tell cre/
: + or cre/cre is by breeding and look at the Mendelian ratio. But if you pay
: enough attention the intensity of PCR genotyping band normally can give you
: some clue that two copy PCR band is much brighter than one copy. Better
: approach is do cre PCR and a control PCR (say a-actin) in one tube, use the
: right cre/+ cre/cre tail DNA control to help you establish the criteria to
: judge what is cre/+ or cre/cre (eg. two band wit
c*l
47 楼
How possible?
You basicly have 2 products in that way. You think your sequencing machine
is sensitive enough to detect one molecule of DNA sequence.
The only possible way is to do a "Pan-PCR" to clone the insertion site which
could actually be very complicated and time-consuming.
Q-PCR is able to distinguish the difference of one and two copies difference
if you have fine pipetting skill.
For most people, just forget about it unless you are having problem breeding
cre/loxp sites together. Then,y
【在 h********n 的大作中提到】
: 我昨天在想这个Cre的问题。 我想， 能不能用这个transgene头围的序列设计primer测
: 序， 测出transgene前后的序列， 然后设计primer根据区分homo hetero and wt.
: homo和wt都是一条带， hetero两条带。
:
: cre/
: pay
: you
: the
: cre,
: different
You basicly have 2 products in that way. You think your sequencing machine
is sensitive enough to detect one molecule of DNA sequence.
The only possible way is to do a "Pan-PCR" to clone the insertion site which
could actually be very complicated and time-consuming.
Q-PCR is able to distinguish the difference of one and two copies difference
if you have fine pipetting skill.
For most people, just forget about it unless you are having problem breeding
cre/loxp sites together. Then,y
【在 h********n 的大作中提到】
: 我昨天在想这个Cre的问题。 我想， 能不能用这个transgene头围的序列设计primer测
: 序， 测出transgene前后的序列， 然后设计primer根据区分homo hetero and wt.
: homo和wt都是一条带， hetero两条带。
:
: cre/
: pay
: you
: the
: cre,
: different
n*k
48 楼
As far as I know, Cre has demonstrated effects in lots of line...while in
general loxP effects shall be controlled easily...if it does, u shall have
hypomoph, right? I don't think ur boss's argument for this particular is
that sound...anyway, people use the other breeding strategy (without control
for cre) all the time. If u have a cell death phenotype, then u need to be
extra careful about cre
【在 D*a 的大作中提到】
: 另外刚跟老板讨论这个问题，如果用cre /cre，cre倒是有control了，可是loxp位点没
: 有control了，也许你加进去的loxp位点somehow影响基因表达呢？所以我们组用的wt
: mice实际上是flox/flox, 作图的时候写"control"，不写"wt"
: 也有人为了这个会多加一个纯粹的wt/wt作为control group, 用来跟flox/flox比较证
: 明flox/flox是wt的表型。
general loxP effects shall be controlled easily...if it does, u shall have
hypomoph, right? I don't think ur boss's argument for this particular is
that sound...anyway, people use the other breeding strategy (without control
for cre) all the time. If u have a cell death phenotype, then u need to be
extra careful about cre
【在 D*a 的大作中提到】
: 另外刚跟老板讨论这个问题，如果用cre /cre，cre倒是有control了，可是loxp位点没
: 有control了，也许你加进去的loxp位点somehow影响基因表达呢？所以我们组用的wt
: mice实际上是flox/flox, 作图的时候写"control"，不写"wt"
: 也有人为了这个会多加一个纯粹的wt/wt作为control group, 用来跟flox/flox比较证
: 明flox/flox是wt的表型。
n*k
49 楼
It is indeed pretty easy to go for another generation of breeding, that way
you know double or single copy of cre and you only need to start with very
few and then get tons of them.., with my own experience, it saves time and
money unless u only need a few animals...
As for to determine the locus, I suppose one could go for FISH like you
point it out...maybe one could kind of go a RACE approach like insertational
mutation genesis(not sure just my guess), or one could even use Parrallel
sequen
【在 c****l 的大作中提到】
: How possible?
: You basicly have 2 products in that way. You think your sequencing machine
: is sensitive enough to detect one molecule of DNA sequence.
: The only possible way is to do a "Pan-PCR" to clone the insertion site which
: could actually be very complicated and time-consuming.
: Q-PCR is able to distinguish the difference of one and two copies difference
: if you have fine pipetting skill.
: For most people, just forget about it unless you are having problem breeding
: cre/loxp sites together. Then,y
you know double or single copy of cre and you only need to start with very
few and then get tons of them.., with my own experience, it saves time and
money unless u only need a few animals...
As for to determine the locus, I suppose one could go for FISH like you
point it out...maybe one could kind of go a RACE approach like insertational
mutation genesis(not sure just my guess), or one could even use Parrallel
sequen
【在 c****l 的大作中提到】
: How possible?
: You basicly have 2 products in that way. You think your sequencing machine
: is sensitive enough to detect one molecule of DNA sequence.
: The only possible way is to do a "Pan-PCR" to clone the insertion site which
: could actually be very complicated and time-consuming.
: Q-PCR is able to distinguish the difference of one and two copies difference
: if you have fine pipetting skill.
: For most people, just forget about it unless you are having problem breeding
: cre/loxp sites together. Then,y
c*l
50 楼
Not as easy as you thought, let's say you will got a litter of ten mice, are
you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
homo Cre? Since the mice are good for breeder only 6 months or so, by the
time you figure out which is homo Cre, you are only left with 3 months. I do
not think it is practical to do this routinely.
RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
cDNA, has nothing to do with insertional site analysis.
not sure about par
【在 n********k 的大作中提到】
: It is indeed pretty easy to go for another generation of breeding, that way
: you know double or single copy of cre and you only need to start with very
: few and then get tons of them.., with my own experience, it saves time and
: money unless u only need a few animals...
: As for to determine the locus, I suppose one could go for FISH like you
: point it out...maybe one could kind of go a RACE approach like insertational
: mutation genesis(not sure just my guess), or one could even use Parrallel
: sequen
you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
homo Cre? Since the mice are good for breeder only 6 months or so, by the
time you figure out which is homo Cre, you are only left with 3 months. I do
not think it is practical to do this routinely.
RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
cDNA, has nothing to do with insertional site analysis.
not sure about par
【在 n********k 的大作中提到】
: It is indeed pretty easy to go for another generation of breeding, that way
: you know double or single copy of cre and you only need to start with very
: few and then get tons of them.., with my own experience, it saves time and
: money unless u only need a few animals...
: As for to determine the locus, I suppose one could go for FISH like you
: point it out...maybe one could kind of go a RACE approach like insertational
: mutation genesis(not sure just my guess), or one could even use Parrallel
: sequen
D*a
51 楼
其实我觉得，一旦发现breeding死活拿不到想要的老鼠，也就差不多确定cre的位置了
吧。。。project也就算是挂了吧。。。也就没必要测cre到底具体在哪儿了吧。。。
如果想要的老鼠频率比较低，那就凑合着用吧。。。
way
insertational
【在 n********k 的大作中提到】
: It is indeed pretty easy to go for another generation of breeding, that way
: you know double or single copy of cre and you only need to start with very
: few and then get tons of them.., with my own experience, it saves time and
: money unless u only need a few animals...
: As for to determine the locus, I suppose one could go for FISH like you
: point it out...maybe one could kind of go a RACE approach like insertational
: mutation genesis(not sure just my guess), or one could even use Parrallel
: sequen
吧。。。project也就算是挂了吧。。。也就没必要测cre到底具体在哪儿了吧。。。
如果想要的老鼠频率比较低，那就凑合着用吧。。。
way
insertational
【在 n********k 的大作中提到】
: It is indeed pretty easy to go for another generation of breeding, that way
: you know double or single copy of cre and you only need to start with very
: few and then get tons of them.., with my own experience, it saves time and
: money unless u only need a few animals...
: As for to determine the locus, I suppose one could go for FISH like you
: point it out...maybe one could kind of go a RACE approach like insertational
: mutation genesis(not sure just my guess), or one could even use Parrallel
: sequen
D*a
52 楼
我觉得不用等3个月，既然小老鼠也不需要，买几只OF1，一窝生十几个的，等三个星期
，生出一窝来把尾巴一剪，一天pcr就出来了
如果是为了breeding用，那就看你需要的老鼠数量了。
假设十只老鼠都是cre+，其中三只是cre+/+, 为了省后续工作，那么等三个星期测cre
也许值得
假如自己就四五只cre，我觉得还是管他纯合杂合，所有cre都放进去breeding，先拿到
后代进行前期实验，之后再看哪只cre纯合来用于批量生产。
是不是可以这样，pcr少P几圈，找个定量的仪器差不多定量下，排除几只肯定是杂合的
就是了，剩下不能确定的去breeding等三个星期。
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
，生出一窝来把尾巴一剪，一天pcr就出来了
如果是为了breeding用，那就看你需要的老鼠数量了。
假设十只老鼠都是cre+，其中三只是cre+/+, 为了省后续工作，那么等三个星期测cre
也许值得
假如自己就四五只cre，我觉得还是管他纯合杂合，所有cre都放进去breeding，先拿到
后代进行前期实验，之后再看哪只cre纯合来用于批量生产。
是不是可以这样，pcr少P几圈，找个定量的仪器差不多定量下，排除几只肯定是杂合的
就是了，剩下不能确定的去breeding等三个星期。
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
h*n
53 楼
Can I just use genotyping primer to run qPCR on genomic DNA?
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
c*l
54 楼
why not. check out sybr green. make sure no non-specific band or primer
dimer which will interfere with your Q-PCR readout. Usually the Q-PCR
machine will do a dissociation curve.
【在 h********n 的大作中提到】
: Can I just use genotyping primer to run qPCR on genomic DNA?
:
: are
: is
: do
dimer which will interfere with your Q-PCR readout. Usually the Q-PCR
machine will do a dissociation curve.
【在 h********n 的大作中提到】
: Can I just use genotyping primer to run qPCR on genomic DNA?
:
: are
: is
: do
n*k
55 楼
sure, I know RACE, that's why I said kind of like RACE...
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
are
is
do
【在 c****l 的大作中提到】
: Not as easy as you thought, let's say you will got a litter of ten mice, are
: you gonna breed all of the Cre+ mice with Cre- mice to figure out which is
: homo Cre? Since the mice are good for breeder only 6 months or so, by the
: time you figure out which is homo Cre, you are only left with 3 months. I do
: not think it is practical to do this routinely.
: RACE, is Rapid Amplification of cDNA Ends which is used to get full-lenght
: cDNA, has nothing to do with insertional site analysis.
: not sure about par
h*n
56 楼
为啥测序这么难呢? 如果我只要知道Cre插入的前后800bp的序列, 也这么难吗?
谢谢.
which
difference
breeding
roughtly
【在 c****l 的大作中提到】
: How possible?
: You basicly have 2 products in that way. You think your sequencing machine
: is sensitive enough to detect one molecule of DNA sequence.
: The only possible way is to do a "Pan-PCR" to clone the insertion site which
: could actually be very complicated and time-consuming.
: Q-PCR is able to distinguish the difference of one and two copies difference
: if you have fine pipetting skill.
: For most people, just forget about it unless you are having problem breeding
: cre/loxp sites together. Then,y
谢谢.
which
difference
breeding
roughtly
【在 c****l 的大作中提到】
: How possible?
: You basicly have 2 products in that way. You think your sequencing machine
: is sensitive enough to detect one molecule of DNA sequence.
: The only possible way is to do a "Pan-PCR" to clone the insertion site which
: could actually be very complicated and time-consuming.
: Q-PCR is able to distinguish the difference of one and two copies difference
: if you have fine pipetting skill.
: For most people, just forget about it unless you are having problem breeding
: cre/loxp sites together. Then,y
c*l
57 楼
tell me how do you figure out Cre插入的前后800bp的序列 or even 1 bp up or downstream?
real deal project!
【在 h********n 的大作中提到】
: 为啥测序这么难呢? 如果我只要知道Cre插入的前后800bp的序列, 也这么难吗?
: 谢谢.
:
: which
: difference
: breeding
: roughtly
real deal project!
【在 h********n 的大作中提到】
: 为啥测序这么难呢? 如果我只要知道Cre插入的前后800bp的序列, 也这么难吗?
: 谢谢.
:
: which
: difference
: breeding
: roughtly
h*n
58 楼
设计primer跟插入片段头尾分别互补, 然后分别往上和往下走, 产物测序?
downstream?
【在 c****l 的大作中提到】
: tell me how do you figure out Cre插入的前后800bp的序列 or even 1 bp up or downstream?
: real deal project!
downstream?
【在 c****l 的大作中提到】
: tell me how do you figure out Cre插入的前后800bp的序列 or even 1 bp up or downstream?
: real deal project!
D*a
59 楼
你搞清楚cre在哪里干什么？能发paper么？。。。
c*l
60 楼
How many copies do you think you will get by that way? Is that enough for
sequencing?
Don't you need a PAIR of primers on each side of your inserted transgene?
Without knowing the upstream or downstream sequence of your integration site
, how do you design the primer outside of your transgene?
I start doubting you really understand what is PCR.
【在 h********n 的大作中提到】
: 设计primer跟插入片段头尾分别互补, 然后分别往上和往下走, 产物测序?
:
: downstream?
sequencing?
Don't you need a PAIR of primers on each side of your inserted transgene?
Without knowing the upstream or downstream sequence of your integration site
, how do you design the primer outside of your transgene?
I start doubting you really understand what is PCR.
【在 h********n 的大作中提到】
: 设计primer跟插入片段头尾分别互补, 然后分别往上和往下走, 产物测序?
:
: downstream?
c*l
61 楼
Sometimes, like in my case, homo cre cause neurological symptom, do you want
to figure out which gene is disrupted? Could be a potential CNS paper. :-)
【在 D*a 的大作中提到】
: 你搞清楚cre在哪里干什么？能发paper么？。。。
to figure out which gene is disrupted? Could be a potential CNS paper. :-)
【在 D*a 的大作中提到】
: 你搞清楚cre在哪里干什么？能发paper么？。。。
D*a
62 楼
这种情况下倒是不错啊 ;D
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
h*n
63 楼
I mean sequencing primer.
when you design primer to sequence sth, you don't need a pair of primers. So
if I design a primer from the head of insertion and go upstream to sequence
the unknown, I only need to know 100 bp sequence. then based on this 100 bp
I can design primer, one primer on transgene and one on upstream, whose
product is cross the insertion starting point, so to distinguish homo vs
hetero.
But I don't know whether the sequencing pcr can be done on genomic DNA.
BTW, when you doubt
【在 c****l 的大作中提到】
: How many copies do you think you will get by that way? Is that enough for
: sequencing?
: Don't you need a PAIR of primers on each side of your inserted transgene?
: Without knowing the upstream or downstream sequence of your integration site
: , how do you design the primer outside of your transgene?
: I start doubting you really understand what is PCR.
when you design primer to sequence sth, you don't need a pair of primers. So
if I design a primer from the head of insertion and go upstream to sequence
the unknown, I only need to know 100 bp sequence. then based on this 100 bp
I can design primer, one primer on transgene and one on upstream, whose
product is cross the insertion starting point, so to distinguish homo vs
hetero.
But I don't know whether the sequencing pcr can be done on genomic DNA.
BTW, when you doubt
【在 c****l 的大作中提到】
: How many copies do you think you will get by that way? Is that enough for
: sequencing?
: Don't you need a PAIR of primers on each side of your inserted transgene?
: Without knowing the upstream or downstream sequence of your integration site
: , how do you design the primer outside of your transgene?
: I start doubting you really understand what is PCR.
h*n
64 楼
I am thinking of running a microarray, comparing gene expression profile on
beta-actin-Cre homo, hetero and wt mice.
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
beta-actin-Cre homo, hetero and wt mice.
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
c*l
65 楼
The sequencing you mentioned needs the DNA to be certain concentration,
definitely not a few copies. Don't you need do a PCR before sequencing? What
is that step for? To increase your template copies!
So
sequence
bp
【在 h********n 的大作中提到】
: I mean sequencing primer.
: when you design primer to sequence sth, you don't need a pair of primers. So
: if I design a primer from the head of insertion and go upstream to sequence
: the unknown, I only need to know 100 bp sequence. then based on this 100 bp
: I can design primer, one primer on transgene and one on upstream, whose
: product is cross the insertion starting point, so to distinguish homo vs
: hetero.
: But I don't know whether the sequencing pcr can be done on genomic DNA.
: BTW, when you doubt
definitely not a few copies. Don't you need do a PCR before sequencing? What
is that step for? To increase your template copies!
So
sequence
bp
【在 h********n 的大作中提到】
: I mean sequencing primer.
: when you design primer to sequence sth, you don't need a pair of primers. So
: if I design a primer from the head of insertion and go upstream to sequence
: the unknown, I only need to know 100 bp sequence. then based on this 100 bp
: I can design primer, one primer on transgene and one on upstream, whose
: product is cross the insertion starting point, so to distinguish homo vs
: hetero.
: But I don't know whether the sequencing pcr can be done on genomic DNA.
: BTW, when you doubt
n*k
66 楼
that's why I suggest to kind of do a RACE approach, first priming with a Cre
primer, and then do similiar like
RACE...I don't know but I want to believe there is a way to do this since
the insertional mutagenesis has been
done, in a way, it has the same difficulity to determine the location as Cre
transgene...
What
【在 c****l 的大作中提到】
: The sequencing you mentioned needs the DNA to be certain concentration,
: definitely not a few copies. Don't you need do a PCR before sequencing? What
: is that step for? To increase your template copies!
:
: So
: sequence
: bp
primer, and then do similiar like
RACE...I don't know but I want to believe there is a way to do this since
the insertional mutagenesis has been
done, in a way, it has the same difficulity to determine the location as Cre
transgene...
What
【在 c****l 的大作中提到】
: The sequencing you mentioned needs the DNA to be certain concentration,
: definitely not a few copies. Don't you need do a PCR before sequencing? What
: is that step for? To increase your template copies!
:
: So
: sequence
: bp
c*l
67 楼
Are you talking about 5' RACE to let the product ligate into a loop? then
you will be able to design anther primer using known sequence. However in
the case of a transgene, it is no as simple as 5'RACE since very often the
transgene construct forms concatemers which greatly increase the complexity
of real products. there are several of these similar techniques such as "Pan
PCR", "selective circularization" or sth etc.
There are also commercial kits available.
Cre
Cre
【在 n********k 的大作中提到】
: that's why I suggest to kind of do a RACE approach, first priming with a Cre
: primer, and then do similiar like
: RACE...I don't know but I want to believe there is a way to do this since
: the insertional mutagenesis has been
: done, in a way, it has the same difficulity to determine the location as Cre
: transgene...
:
: What
you will be able to design anther primer using known sequence. However in
the case of a transgene, it is no as simple as 5'RACE since very often the
transgene construct forms concatemers which greatly increase the complexity
of real products. there are several of these similar techniques such as "Pan
PCR", "selective circularization" or sth etc.
There are also commercial kits available.
Cre
Cre
【在 n********k 的大作中提到】
: that's why I suggest to kind of do a RACE approach, first priming with a Cre
: primer, and then do similiar like
: RACE...I don't know but I want to believe there is a way to do this since
: the insertional mutagenesis has been
: done, in a way, it has the same difficulity to determine the location as Cre
: transgene...
:
: What
n*k
68 楼
thanks, I c....so I guess that's not the case for insertional mutagenesis,
then? I see your points, however, still can the concatemers problem be
overcame with introducing an extra cloning step with primers designed
containing specific rare enzyme cut sites...
complexity
Pan
【在 c****l 的大作中提到】
: Are you talking about 5' RACE to let the product ligate into a loop? then
: you will be able to design anther primer using known sequence. However in
: the case of a transgene, it is no as simple as 5'RACE since very often the
: transgene construct forms concatemers which greatly increase the complexity
: of real products. there are several of these similar techniques such as "Pan
: PCR", "selective circularization" or sth etc.
: There are also commercial kits available.
:
: Cre
: Cre
then? I see your points, however, still can the concatemers problem be
overcame with introducing an extra cloning step with primers designed
containing specific rare enzyme cut sites...
complexity
Pan
【在 c****l 的大作中提到】
: Are you talking about 5' RACE to let the product ligate into a loop? then
: you will be able to design anther primer using known sequence. However in
: the case of a transgene, it is no as simple as 5'RACE since very often the
: transgene construct forms concatemers which greatly increase the complexity
: of real products. there are several of these similar techniques such as "Pan
: PCR", "selective circularization" or sth etc.
: There are also commercial kits available.
:
: Cre
: Cre
c*l
69 楼
You can try. But you have to have restriction site inside your construct and
either 5' upstream or 3' downstream and not too far from your insertion
sites (say not more than a couple of kb. the bigger the size of your product
, the lower the efficiency of PCR)
In the simplest situation, say the concatemers are all head to tail and only
one restriction site in the construct and it is very close to your
insertion sites in the genomic DNA, you will still have 2 products.
【在 n********k 的大作中提到】
: thanks, I c....so I guess that's not the case for insertional mutagenesis,
: then? I see your points, however, still can the concatemers problem be
: overcame with introducing an extra cloning step with primers designed
: containing specific rare enzyme cut sites...
:
: complexity
: Pan
either 5' upstream or 3' downstream and not too far from your insertion
sites (say not more than a couple of kb. the bigger the size of your product
, the lower the efficiency of PCR)
In the simplest situation, say the concatemers are all head to tail and only
one restriction site in the construct and it is very close to your
insertion sites in the genomic DNA, you will still have 2 products.
【在 n********k 的大作中提到】
: thanks, I c....so I guess that's not the case for insertional mutagenesis,
: then? I see your points, however, still can the concatemers problem be
: overcame with introducing an extra cloning step with primers designed
: containing specific rare enzyme cut sites...
:
: complexity
: Pan
D*a
70 楼
想到下面几个问题:
cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
基因组不同
有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
测多长的片段可以确定说是这个位点？
cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
基因组不同
有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
测多长的片段可以确定说是这个位点？
D*a
71 楼
曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
来找的。
不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
感兴趣我回头找找
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
来找的。
不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
感兴趣我回头找找
want
【在 c****l 的大作中提到】
: Sometimes, like in my case, homo cre cause neurological symptom, do you want
: to figure out which gene is disrupted? Could be a potential CNS paper. :-)
n*k
72 楼
good points...nonetheless, for the most, this won't be a concern and if you
look at the whole genome mutagenesis in 129 or alike, they just sequence
about 100bp or even 30 bp, enough to determine the locus for most...
background
【在 D*a 的大作中提到】
: 想到下面几个问题:
: cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
: 基因组不同
: 有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
: 测多长的片段可以确定说是这个位点？
look at the whole genome mutagenesis in 129 or alike, they just sequence
about 100bp or even 30 bp, enough to determine the locus for most...
background
【在 D*a 的大作中提到】
: 想到下面几个问题:
: cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
: 基因组不同
: 有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
: 测多长的片段可以确定说是这个位点？
n*k
73 楼
this is very classical genome mapping...those ENU people do this all the
time before genome era and still does quite a bit too...
【在 D*a 的大作中提到】
: 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
: 鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
: 来找的。
: 不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
: 感兴趣我回头找找
:
: want
time before genome era and still does quite a bit too...
【在 D*a 的大作中提到】
: 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
: 鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
: 来找的。
: 不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
: 感兴趣我回头找找
:
: want
c*l
74 楼
I am interested but sure if the approach you mention will be suitable for my
purpose. Pls let me know the principle at least. Thanks.
【在 D*a 的大作中提到】
: 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
: 鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
: 来找的。
: 不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
: 感兴趣我回头找找
:
: want
purpose. Pls let me know the principle at least. Thanks.
【在 D*a 的大作中提到】
: 曾经为了考试，做过一个寻找mutation的project作业，用的是两只不同background老
: 鼠交配，然后通过pcr不同background的Polymorphism的marker，来计算后代的重组率
: 来找的。
: 不知道能不能用在这里，是什么的Polymorphism已经忘了。。。snp还是微卫星？你要
: 感兴趣我回头找找
:
: want
D*a
75 楼
thanks, 请问对照有软件什么的么？还是手工去数据库比？
you
【在 n********k 的大作中提到】
: good points...nonetheless, for the most, this won't be a concern and if you
: look at the whole genome mutagenesis in 129 or alike, they just sequence
: about 100bp or even 30 bp, enough to determine the locus for most...
:
: background
you
【在 n********k 的大作中提到】
: good points...nonetheless, for the most, this won't be a concern and if you
: look at the whole genome mutagenesis in 129 or alike, they just sequence
: about 100bp or even 30 bp, enough to determine the locus for most...
:
: background
d*0
76 楼
feels like virtually impossible. I had discussion on similar operation long
time ago with some expert, he told me it would be very hard, if not
impposible, to get your positive line. just dont remember the detail he gave
.
time ago with some expert, he told me it would be very hard, if not
impposible, to get your positive line. just dont remember the detail he gave
.
D*a
77 楼
用的是QTL，quantitative trait loci
pubmed搜qtl mapping 可以搜出来一堆
找了找我上课的几张图，看看能不能发到你邮箱
工作量很大啊。。整天pcr好无聊
my
【在 c****l 的大作中提到】
: I am interested but sure if the approach you mention will be suitable for my
: purpose. Pls let me know the principle at least. Thanks.
pubmed搜qtl mapping 可以搜出来一堆
找了找我上课的几张图，看看能不能发到你邮箱
工作量很大啊。。整天pcr好无聊
my
【在 c****l 的大作中提到】
: I am interested but sure if the approach you mention will be suitable for my
: purpose. Pls let me know the principle at least. Thanks.
c*l
78 楼
Thank you very much. I understand QTL, very traditional gene mapping
approach. Do we really have to to this in a post-genomic era plus we know
the insertion sequence?
【在 D*a 的大作中提到】
: 用的是QTL，quantitative trait loci
: pubmed搜qtl mapping 可以搜出来一堆
: 找了找我上课的几张图，看看能不能发到你邮箱
: 工作量很大啊。。整天pcr好无聊
:
: my
approach. Do we really have to to this in a post-genomic era plus we know
the insertion sequence?
【在 D*a 的大作中提到】
: 用的是QTL，quantitative trait loci
: pubmed搜qtl mapping 可以搜出来一堆
: 找了找我上课的几张图，看看能不能发到你邮箱
: 工作量很大啊。。整天pcr好无聊
:
: my
D*a
79 楼
如果ls几位说的测序能做的话还是测序快吧
很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
【在 c****l 的大作中提到】
: Thank you very much. I understand QTL, very traditional gene mapping
: approach. Do we really have to to this in a post-genomic era plus we know
: the insertion sequence?
很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
【在 c****l 的大作中提到】
: Thank you very much. I understand QTL, very traditional gene mapping
: approach. Do we really have to to this in a post-genomic era plus we know
: the insertion sequence?
h*n
80 楼
design primer for homo vs hetero?
background
【在 D*a 的大作中提到】
: 想到下面几个问题:
: cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
: 基因组不同
: 有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
: 测多长的片段可以确定说是这个位点？
background
【在 D*a 的大作中提到】
: 想到下面几个问题:
: cre周围的序列测出来之后怎么办？现在有老鼠全基因组图了？别忘了不同background
: 基因组不同
: 有计算机工具可以通过一段序列搜索全基因组（或者某染色体）来对比么？
: 测多长的片段可以确定说是这个位点？
h*n
81 楼
I agree, spend some money and get sequence.
【在 D*a 的大作中提到】
: 如果ls几位说的测序能做的话还是测序快吧
: 很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
【在 D*a 的大作中提到】
: 如果ls几位说的测序能做的话还是测序快吧
: 很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
c*l
82 楼
Do you mean whole genome sequencing, r u insane?
【在 h********n 的大作中提到】
: I agree, spend some money and get sequence.
【在 h********n 的大作中提到】
: I agree, spend some money and get sequence.
c*l
83 楼
How do you sequence? Where to start? I have spent so many posts on this, no
easy way sequencing unless you are talking about whole genome sequencing.
【在 D*a 的大作中提到】
: 如果ls几位说的测序能做的话还是测序快吧
: 很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
easy way sequencing unless you are talking about whole genome sequencing.
【在 D*a 的大作中提到】
: 如果ls几位说的测序能做的话还是测序快吧
: 很久没有追踪这方面的进展了，我也不知道发展到什么程度了:)
c*o
84 楼
即使是一个copy的Cre转基因也应该有control。记得nature发过一篇关于Cre的
toxicity的paper，大致是因为很多转基因的Cre都是很强的promoter驱动的，会影响插
入位点附近的基因表达。所以floxed/floxed并不是floxed/floxed;Cre的最好control
，应该是floxed/wt,Cre。
现在有一些cre是knockin在promoter下游的，至少位置是清楚的。另外一个办法是
knockin在Rosa26的位置。
Homo Cre有phenotype是挺有意思的，原来隔壁实验室就有过，但花了很多时间想搞清
楚插入位点，但最后是不了了之了。
toxicity的paper，大致是因为很多转基因的Cre都是很强的promoter驱动的，会影响插
入位点附近的基因表达。所以floxed/floxed并不是floxed/floxed;Cre的最好control
，应该是floxed/wt,Cre。
现在有一些cre是knockin在promoter下游的，至少位置是清楚的。另外一个办法是
knockin在Rosa26的位置。
Homo Cre有phenotype是挺有意思的，原来隔壁实验室就有过，但花了很多时间想搞清
楚插入位点，但最后是不了了之了。
h*n
85 楼
not whole genome. I am talking with the facility and try to have them work
for me.
【在 c****l 的大作中提到】
: Do you mean whole genome sequencing, r u insane?
for me.
【在 c****l 的大作中提到】
: Do you mean whole genome sequencing, r u insane?
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