Chromatin Immunoprecipitation (for E. coli)

Chromatin Immunoprecipitation (for E. coli)
Chromatin Immunoprecipitation (for E. coli)

Chromatin Immunoprecipitation (for E. coli)

Making crosslinked, sonicated cell lysates (“chromatin”)

1. Grow 2 ml o/n culture. Subculture 1/100 (e.g. 400 μl à 40 ml). Grow to appropriate OD600 (typically 0.3-0.6).

2. Crosslink cells by adding formaldehyde to final concentration of 1% (~1 ml per 40 ml culture). Swirl flask to mix. Incubate for 20 min at room temperature.

3. Quench formaldehyde by adding glycine to final concentration of 0.5 M (typically 10 ml of 2.5 M glycine per 40 ml culture).

4. Spin cells at full speed in bench-top centrifuge. Resuspend in TBS (original culture volume). Spin again. Resuspend in 1 ml TBS. Spin in microcentrfuge, max speed, 1 min. Discard supernatant. Cells can be frozen at this point.

5. Resuspend cells in 1 ml FA lysis buffer containing 2 mg/ml lysoszyme. Incubate @ 37 °C for 30 min to lyse cells. Crosslinked lysates can be frozen at this point.

6. Chill on ice for >5 min. Sonicate to shear DNA: 2 x 30 s pulses, 50% output, keeping cells on ice. In between 30 s pulses leave cells on ice for >3 minutes.

7. Spin cell lysates in microcentrifuge, max speed, 5 min. Keep supernatant (should be ~1 ml). For cultures >20 ml, dilute crosslinked lysates to 1 ml per 20 ml original culture volume with FA lysis buffer, e.g. if original culture volume was 40 ml, dilute crosslinked cell lysates to final volume of 2 ml. Crosslinked, sonicated lysates can be frozen at this point.

Immunoprecipitation (with protein A beads)

1. Dilute cross linked, sonicated lysate in FA lysis buffer such that there is 800 μl for every 10 ml original culture volume (assuming exponential phase growth).

2. Add 25 μl protein A beads (from 50% slurry in TBS) and appropriate amount of antibody. Incubate for 90 min at room temperature on rotisserie rotator.

3. Spin IP mix for 1 minute in microcentrifuge at 4,000 rpm. Remove supernatant by aspiration. Resuspend in 750 μl FA lysis buffer and transfer to Spin-X column filter.

4. Incubate for 3 min at room temperature on rotisserie rotator in Spin-X column. Spin (as before) and discard supernatant. Repeat wash step with FA lysis buffer, FA lysis buffer + 500 mM NaCl, ChIP wash buffer, and TE (10 mM Tris pH 8.0, 1 mM EDTA).

5. Resuspend beads in 100 μl ChIP elution buffer. Place Spin-X filter in fresh dolphin-nosed tube.

Incubate for 10 min at 65 °C with occasional gentle mixing. Spin for 1 minute in microcentrifuge at 4,000 rpm. Discard filter.

6. Decrosslink by incubating o/n at 65 °C or 10 min at 100 °C (in normal 1.5 ml tube). Decrosslink input sample (i.e. crosslinked, sonicated cell lysate used for IP; typically 20 μl) as necessary.

7. Clean up DNA with Qiagen PCR purification kit. Use 2 μl of eluted DNA (typically from 200 μl) per 10 μl qPCR reaction.

Immunoprecipitation (with IgG beads – for TAP-tagged proteins)

1. Dilute crosslinked, sonicated lysate in FA lysis buffer such that there is 800 μl for every 10 ml original culture volume (assuming exponential phase growth).

2. Add 25 μl IgG beads (from 50% slurry in TBS). Incubate for 90 min at room temperature on rotisserie rotator. Continue from step 3 above.

Buffers

FA lysis buffer (150/500 mM NaCl)

50 mM Hepes-KOH, pH 7. 150/500 mM NaCl (use low salt buffer for all steps except for one wash step)

1 mM EDTA 1% Triton X-100 0.1% sodium deoxycholate 0.1% SDS

ChIP wash buffer

10 mM Tris-HCl, pH 8.0 250 mM LiCl 1 mM EDTA 0.5% Nonidet-P40

0.5% sodium deoxycholate ChIP elution buffer 50 mM Tris-HCl, pH 7.5

10 mM EDTA 1% SDS

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lovestory歌词中英文对照

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(完整版)介词for用法归纳

介词for用法归纳 用法1:(表目的)为了。如: They went out for a walk. 他们出去散步了。 What did you do that for? 你干吗这样做? That’s what we’re here for. 这正是我们来的目的。 What’s she gone for this time? 她这次去干什么去了? He was waiting for the bus. 他在等公共汽车。 【用法说明】在通常情况下,英语不用for doing sth 来表示目的。如: 他去那儿看他叔叔。 误:He went there for seeing his uncle. 正:He went there to see his uncle. 但是,若一个动名词已名词化,则可与for 连用表目的。如: He went there for swimming. 他去那儿游泳。(swimming 已名词化) 注意:若不是表目的,而是表原因、用途等,则其后可接动名词。(见下面的有关用法) 用法2:(表利益)为,为了。如: What can I do for you? 你想要我什么? We study hard for our motherland. 我们为祖国努力学习。 Would you please carry this for me? 请你替我提这个东西好吗? Do more exercise for the good of your health. 为了健康你要多运动。 【用法说明】(1) 有些后接双宾语的动词(如buy, choose, cook, fetch, find, get, order, prepare, sing, spare 等),当双宾语易位时,通常用for 来引出间接宾语,表示间接宾语为受益者。如: She made her daughter a dress. / She made a dress for her daughter. 她为她女儿做了件连衣裙。 He cooked us some potatoes. / He cooked some potatoes for us. 他为我们煮了些土豆。 注意,类似下面这样的句子必须用for: He bought a new chair for the office. 他为办公室买了张新办公椅。 (2) 注意不要按汉语字面意思,在一些及物动词后误加介词for: 他们决定在电视上为他们的新产品打广告。 误:They decided to advertise for their new product on TV. 正:They decided to advertise their new product on TV. 注:advertise 可用作及物或不及物动词,但含义不同:advertise sth=为卖出某物而打广告;advertise for sth=为寻找某物而打广告。如:advertise for a job=登广告求职。由于受汉语“为”的影响,而此处误加了介词for。类似地,汉语中的“为人民服务”,说成英语是serve the people,而不是serve for the people,“为某人的死报仇”,说成英语是avenge sb’s death,而不是avenge for sb’s death,等等。用法3:(表用途)用于,用来。如: Knives are used for cutting things. 小刀是用来切东西的。 This knife is for cutting bread. 这把小刀是用于切面包的。 It’s a machine for slicing bread. 这是切面包的机器。 The doctor gave her some medicine for her cold. 医生给了她一些感冒药。 用法4:为得到,为拿到,为取得。如: He went home for his book. 他回家拿书。 He went to his friend for advice. 他去向朋友请教。 She often asked her parents for money. 她经常向父母要钱。

(完整版)SeasonsintheSun_中文歌词翻译_中英对照

Seasons in the Sun_中文歌词翻译_中英对照 goodbye to you my trusted friend.再见了,我忠实的朋友. we're known each other we're 9 or 10.我们从孩提时就已相识相知. together we've climb hills trees.我们一起爬山上树. learned of love abc.一起学习. skinned our hearts skinned our knees.我们心意相通,情如手足. goodbye my friend it's hard to die.再见了,朋友们,我本不愿离去. when all the birds are singing in the sky.当所有的鸟儿在天空歌唱. now the spring is in the air.空气中弥漫着春天的气息. pretty girls are everywhere.到处是漂亮女孩. think of me and i'll be there.想我,我便与你同在. we had joy,we had fun.我们曾如此快乐. we had seasons in the sun.也曾充满阳光. but the hills that we climbed were just seasons out of time.那些日子已然逝去. goodbye papa please pray for me.再见了爸爸,请为我祈祷. i was the black sheep of the family.我是家里的害群之马. u tried 2 teach me right from wrong.你费尽心思教我明辨是非. too much wine too much song.我却沉醉于歌酒狂欢. wonder how i got along.真不知那些日子是如何度过. goodbye papa is hard 2 die.再见了爸爸,我本不愿离去. when all the birds are singing in the sky.当所有的鸟儿在天空歌唱. now the spring in the air.空气中弥漫着春天的气息. little children everywhere.孩子们到处嬉戏. when u see them i'll be there.当你看见他们,我便会与你同在. we had joy,we had fun.我们曾如此快乐. we had seasons in the sun.也曾有阳光季节. but the wild the song.但昔日的歌酒狂欢. like the season has all gone.犹如季节更迭已消逝. we had joy,we had fun.我们曾如此快乐. we had seasons in the sun.也曾有阳光季节. but the wild the song.但昔日的歌酒狂欢. like the season has all gone.犹如季节更迭已消逝. goodbye michelle my little one.再见了蜜雪儿,我的贝比. u gave me love help me find the sun.你给我爱,给我希望. and every time that i was down.当我意志消沉时.

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英语介词for的用法归纳总结.doc

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Do more exercise for the good of your health. 为了健康你要多运动。 【用法说明】(1) 有些后接双宾语的动词(如buy, choose, cook, fetch, find, get, order, prepare, sing, spare 等),当双宾语易位时,通常用for 来引出间接宾语,表示间接宾语为受益者 She made her daughter a dress. / She made a dress for her daughter. 她为她女儿做了件连衣裙。 He cooked us some potatoes. / He cooked some potatoes for us. 他为我们煮了些土豆。 注意,类似下面这样的句子必须用for: He bought a new chair for the office. 他为办公室买了张新办公椅。 (2) 注意不要按汉语字面意思,在一些及物动词后误加介词for: 他们决定在电视上为他们的新产品打广告。 误:They decided to advertise for their new product on TV. 正:They decided to advertise their new product on TV. 注:advertise 可用作及物或不及物动词,但含义不同:advertise sth=为卖出某物而打广告;advertise for sth=为寻找某物而打广告advertise for a job=登广告求职。由于受汉语为的影响,而此处误加了介词for。类似地,汉语中的为人民服务,说成英语是serve the people,而不是serve for the people,为某人的死报仇,说成英语是avenge sb s death,而不是avenge for sb s death,等等。 用法3:(介词for表用途)用于,用来 Knives are used for cutting things. 小刀是用来切东西的。

介词for用法完全归纳

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seasonsinthesun_中文歌词翻译_中英对照

Seasons?in?the?Sun_中文歌词翻译_中英对照 goodbye to you my trusted friend.再见了,我忠实的朋友. we're known each other we're 9 or 10.我们从孩提时就已相识相知. together we've climb hills trees.我们一起爬山上树. learned of love abc.一起学习. skinned our hearts skinned our knees.我们心意相通,情如手足. goodbye my friend it's hard to die.再见了,朋友们,我本不愿离去. when all the birds are singing in the sky.当所有的鸟儿在天空歌唱. now the spring is in the air.空气中弥漫着春天的气息. pretty girls are everywhere.到处是漂亮女孩. think of me and i'll be there.想我,我便与你同在. we had joy,we had fun.我们曾如此快乐. we had seasons in the sun.也曾充满阳光. but the hills that we climbed were just seasons out of time.那些日子已然逝去. goodbye papa please pray for me.再见了爸爸,请为我祈祷. i was the black sheep of the family.我是家里的害群之马. u tried 2 teach me right from wrong.你费尽心思教我明辨是非. too much wine too much song.我却沉醉于歌酒狂欢. wonder how i got along.真不知那些日子是如何度过. goodbye papa is hard 2 die.再见了爸爸,我本不愿离去. when all the birds are singing in the sky.当所有的鸟儿在天空歌唱. now the spring in the air.空气中弥漫着春天的气息. little children everywhere.孩子们到处嬉戏. when u see them i'll be there.当你看见他们,我便会与你同在. we had joy,we had fun.我们曾如此快乐. we had seasons in the sun.也曾有阳光季节. but the wild the song.但昔日的歌酒狂欢. like the season has all gone.犹如季节更迭已消逝. we had joy,we had fun.我们曾如此快乐. we had seasons in the sun.也曾有阳光季节. but the wild the song.但昔日的歌酒狂欢. like the season has all gone.犹如季节更迭已消逝. goodbye michelle my little one.再见了蜜雪儿,我的贝比. u gave me love help me find the sun.你给我爱,给我希望. and every time that i was down.当我意志消沉时.

for的用法完全归纳

for的用法完全归纳 用法1:(表目的)为了。如: They went out for a walk. 他们出去散步了。 What did you do that for? 你干吗这样做? That’s what we’re here for. 这正是我们来的目的。 What’s she gone for this time? 她这次去干什么去了? He was waiting for the bus. 他在等公共汽车。 在通常情况下,英语不用for doing sth 来表示目的。如:他去那儿看他叔叔。 误:He went there for seeing his uncle.正:He went there to see his uncle. 但是,若一个动名词已名词化,则可与for 连用表目的。如: He went there for swimming. 他去那儿游泳。(swimming 已名词化) 注意:若不是表目的,而是表原因、用途等,则其后可接动名词。 用法2:(表利益)为,为了。如: What can I do for you? 你想要我什么? We study hard for our motherland. 我们为祖国努力学习。 Would you please carry this for me? 请你替我提这个东西好吗? Do more exercise for the good of your health. 为了健康你要多运动。 (1)有些后接双宾语的动词(如buy, choose, cook, fetch, find, get, order, prepare, sing, spare 等),当双宾语易位时,通 常用for 来引出间接宾语,表示间接宾语为受益者。如: She made her daughter a dress. / She made a dress for her daughter. 她为她女儿做了件连衣裙。 He cooked us some potatoes. / He cooked some potatoes for us. 他为我们煮了些土豆。 注意,类似下面这样的句子必须用for: He bought a new chair for the office. 他为办公室买了张新办公椅。 (2) 注意不要按汉语字面意思,在一些及物动词后误加介词for: 他们决定在电视上为他们的新产品打广告。 误:They decided to advertise for their new product on TV. 正:They decided to advertise their new product on TV. 注:advertise 可用作及物或不及物动词,但含义不同:advertise sth=为卖出某物而打广告;advertise for sth=为寻找某物而打广告。如:advertise for a job=登广告求职。由于受汉语“为”的影响,而此处误加了介词for。类似地,汉语中的“为人民服务”,说成英语是serve the people,而不是serve for the people,“为某人的死报仇”,说成英语是avenge sb’s death,而不是avenge for sb’s death,等等。 用法3:(表用途)用于,用来。如: Knives are used for cutting things. 小刀是用来切东西的。 This knife is for cutting bread. 这把小刀是用于切面包的。 It’s a machine for slicing bread. 这是切面包的机器。 The doctor gave her some medicine for her cold. 医生给了她一些感冒药。 用法4:为得到,为拿到,为取得。如: He went home for his book. 他回家拿书。 He went to his friend for advice. 他去向朋友请教。 She often asked her parents for money. 她经常向父母要钱。 We all hope for success. 我们都盼望成功。 Are you coming in for some tea? 你要不要进来喝点茶? 用法5:给(某人),供(某人)用。如: That’s for you. 这是给你的。 Here is a letter for you. 这是你的信。 Have you room for me there? 你那边能给我腾出点地方吗? 用法6:(表原因、理由)因为,由于。如:

歌剧魅影全套歌词中英对照高级翻译超

Turn your face away 把你的脸转向这里 from the garish light of day Night-time sharpe ns 夜色渐浓 heighte ns each sen sati on 知觉萌动 Darkn ess stirs and wakes imag in ati on 冥冥黑暗引领想象出笼 Sile ntly the sen ses aba ndon their defe nces ... 寂静之中感觉幵始放纵 Slowly, gen tly ni ght un furls its sple ndour 夜晚显现魅力缓慢轻柔 Grasp it, sense it - tremulous and tender 抓住它感觉它 颤抖中带着温柔 tur n your thoughts away 把你的心转向这里 from cold, un feeli ng light 脱离那冰冷无情的光 and listen to the music of the night ... 尽情聆听这夜的乐章 Close your eyes and surre nder to your 闭上双眼尽情放纵 darkest dreams! 心灵深处的梦想 不要再看俗气的日光

you knew before! 全部主张! Close your eyes, 闭上双眼 let your spirit start to soar! 让你的灵魂去飞翔! And you'll live 你将拥有新的生活 as you've n ever lived before ... 在你从未生活过的地方 Softly, deftly 不知不觉中 music shall surro und you ... 音乐将你包容 Feel it, hear it, 去感受去聆听 clos ing in aro und you ... 让它进入你的心 Ope n up your mi nd 让你的思想翱翔 let your fan tasies unwind 为你的幻想松绑 in this dark ness which 在这黑暗之中 you know you cannot fight 你知道自己无力抵抗

介词for 的常见用法归纳

介词for 的常见用法归纳 贵州省黔东南州黎平县黎平一中英语组廖钟雁介词for 用法灵活并且搭配能力很强,是一个使用频率非常高的词,也是 高考必考的重要词汇,现将其常见用法归纳如下,供参考。 1.表时间、距离或数量等。 ①意为“在特定时间,定于,安排在约定时间”。如: The meeting is arranged for 9 o’clock. 会议安排在九点进行。 ②意为“持续达”,常于last、stay 、wait等持续性动词连用,表动作持续的时间,有时可以省略。如: He stayed for a long time. 他逗留了很久。 The meeting lasted (for)three hours. 会议持续了三小时。 ③意为“(距离或数量)计、达”。例如: He walked for two miles. 他走了两英里。 The shop sent me a bill for $100.商店给我送来了100美元的账单。 2. 表方向。意为“向、朝、开往、前往”。常与head、leave 、set off、start 等动词连用。如: Tomorrow Tom will leave for Beijing. 明天汤姆要去北京。 He put on his coat and headed for the door他穿上大衣向门口走去。 介词to也可表示方向,但往往与come、drive 、fly、get、go、lead、march、move、return、ride、travel、walk等动词连用。 3.表示理由或原因,意为“因为、由于”。常与thank、famous、reason 、sake 等词连用。如: Thank you for helping me with my English. 谢谢你帮我学习英语。 For several reasons, I’d rather not meet him. 由于种种原因,我宁可不见他。 The West Lake is famous for its beautiful scenery.西湖因美景而闻名。 4.表示目的,意为“为了、取、买”等。如: Let’s go for a walk. 我们出去散步吧。 I came here for my schoolbag.我来这儿取书包。 He plays the piano for pleasure. 他弹钢琴是为了消遣。 There is no need for anyone to know. 没必要让任何人知道。 5.表示动作的对象或接受者,意为“给、为、对于”。如: Let me pick it up for you. 让我为你捡起来。 Watching TV too much is bad for your health. 看电视太多有害于你的健康。 Here is a letter for you. 这儿有你的一封信。

see-you-again整理歌词中英文对照

see you again It's been a long day without you my friend 没有老友你的陪伴日子真是漫长 And I'll tell you all about it when I see you again 与你重逢之时我会敞开心扉倾诉所有 We've come a long way from where we began 回头凝望我们携手走过漫长的旅程 Oh I'll tell you all about it when I see you again 与你重逢之时我会敞开心扉倾诉所有 When I see you again 与你重逢之时 Damn who knew all the planes we flew 谁会了解我们经历过怎样的旅程 Good things we've been through 谁会了解我们见证过怎样的美好 That I'll be standing right here 我都会在这里 Talking to you about another path 与你聊聊另一种选择的可能 I know we loved to hit the road and laugh 我懂我们都喜欢速度与激情 But something told me that it wouldn't last 但有个声音告诉我这美好并不会永恒 Had to switch up look at things different see the bigger picture 如何才能改变观点用更宏观的视野看这世界 Those were the days hard work forever pays 有付出的日子终有收获的时节 Now I see you in a better place 此刻我看到你走进更加美好的未来 Now I see you in a better place 此刻我看到你走进更加美好的未来 How could we not talk about family when family's all that we got?当家人已是我们唯一的牵绊时我们怎么能忘却最可贵的亲情Everything I went through you were standing there by my side 无论历经怎样的艰难坎坷总有你相伴陪我度过 And now you gonna be with me for the last ride 而今你将陪我走完这最后一段旅程 It's been a long day without you my friend 没有老友你的陪伴日子真是漫长 And I'll tell you all about it when I see you again 与你重逢之时我会敞开心扉倾诉所有 We've come a long way from where we began

英语介词的用法总结

介词的用法 1.表示地点位置的介词 1)at ,in, on, to,for at (1)表示在小地方; (2)表示“在……附近,旁边” in (1)表示在大地方; (2)表示“在…范围之内”。 on 表示毗邻,接壤,“在……上面”。 to 表示在……范围外,不强调是否接壤;或“到……” 2)above, over, on 在……上 above 指在……上方,不强调是否垂直,与below相对; over指垂直的上方,与under相对,但over与物体有一定的空间,不直接接触。 on表示某物体上面并与之接触。 The bird is flying above my head. There is a bridge over the river. He put his watch on the desk. 3)below, under 在……下面 under表示在…正下方 below表示在……下,不一定在正下方 There is a cat under the table. Please write your name below the line. 4)in front [frant]of, in the front of在……前面 in front of…意思是“在……前面”,指甲物在乙物之前,两者互不包括;其反义词是behind(在……的后面)。There are some flowers in front of the house.(房子前面有些花卉。) in the front of 意思是“在…..的前部”,即甲物在乙物的内部.反义词是at the back of…(在……范围内的后部)。 There is a blackboard in the front of our classroom. 我们的教室前边有一块黑板。 Our teacher stands in the front of the classroom. 我们的老师站在教室前.(老师在教室里) 5)beside,behind beside 表示在……旁边 behind 表示在……后面 2.表示时间的介词 1)in , on,at 在……时 in表示较长时间,如世纪、朝代、时代、年、季节、月及一般(非特指)的早、中、晚等。 如in the 20th century, in the 1950s, in 1989, in summer, in January, in the morning, in one’s life , in one’s thirties等。 on表示具体某一天及其早、中、晚。 如on May 1st, on Monday, on New Year’s Day, on a cold night in January, on a fine morning, on Sunday afternoon等。 at表示某一时刻或较短暂的时间,或泛指圣诞节,复活节等。 如at 3:20, at this time of year, at the beginning of, at the end of …, at the age of …, at Christmas,at night, at noon, at this moment等。 注意:在last, next, this, that, some, every 等词之前一律不用介词。如:We meet every day. 2)in, after 在……之后 “in +段时间”表示将来的一段时间以后; “after+段时间”表示过去的一段时间以后; “after+将来的时间点”表示将来的某一时刻以后。 3)from, since 自从…… from仅说明什么时候开始,不说明某动作或情况持续多久;

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