iGEM 2017 — Team Worldshaper-Wuhan

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# Team:Worldshaper-Wuhan/Notebook

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* [Home](https://2017.igem.org/Team%3AWorldshaper-Wuhan)
* [About](https://2017.igem.org/Team%3AWorldshaper-Wuhan/about-us)
* [Background](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Background)
* Project

  + [Solution and Design](https://2017.igem.org/Team%3AWorldshaper-Wuhan/solution-and-design)
  + [Results](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Results)
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  + [Measurement](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Demonstrate)
  + [Procedure](https://2017.igem.org/Team%3AWorldshaper-Wuhan/procedure)
  + [Notebook](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Notebook)
* [Safety](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Safety)
* [Human Practice](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Human-Practice)
* [Collaboration](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Collaborations)
* [InterLab](https://2017.igem.org/Team%3AWorldshaper-Wuhan/InterLab)
* [Attributions](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Attributions)
* Awards

  + [Achievement](https://2017.igem.org/Team%3AWorldshaper-Wuhan/HP/Gold_Integrated)
  + [Survey Result](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Engagement)
  + [Applied Design](https://2017.igem.org/Team%3AWorldshaper-Wuhan/Applied_Design)
  + [Silver](https://2017.igem.org/Team%3AWorldshaper-Wuhan/HP/Silver)
  + [Gold](https://2017.igem.org/Team%3AWorldshaper-Wuhan/gold)

# Notebook

**MI0000077**

hsa-mir-21

UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA

72bp

1 tgtcgggtag cttatcagac tgatgttgac tgttgaatct catggcaaca ccagtcgatg ggctgtctga ca

Plasmid EGFP-C1

Xho I  CTCGAG

HindIII  ttcgaa

F:  TCACTCGAGTGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGCAACA

R:     TCAAAGCTTTGTCAGACAGCCCATCGACTGGTGTTGCCATGAGATTCAACAGTCAACAT

**BioBrick Suffix**[[edit](http://parts.igem.org/wiki/index.php?title=Help:BioBrick_Prefix_and_Suffix&action=edit&section=4)]

The standard BioBrick suffix is always:

tactagtagcggccgctgcag

g^acgt c

![q.jpg](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e0fdd6839a0d1d7229c6/1509417222426/q.jpg)![q.jpg](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e0fdd6839a0d1d7229c6/1509417222426/q.jpg?format=750w)

![EcoR I](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e11627ef2d4ad32c3e5d/1509417239963/)![EcoR I](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e11627ef2d4ad32c3e5d/1509417239963/?format=300w)

*EcoR I*

![pst I](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e135652dea485edbe11e/1509417278024/e.jpg)![pst I](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e135652dea485edbe11e/1509417278024/e.jpg?format=300w)

*pst I*

![r.jpg](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e169e4966b2de095fd38/1509417329382/r.jpg)![r.jpg](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e169e4966b2de095fd38/1509417329382/r.jpg?format=750w)

![](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e18527ef2d4ad32c484c/1509417350721/)![](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e18527ef2d4ad32c484c/1509417350721/?format=500w)

miR-21 bulged Renilla luciferase reporter, 6 sites; CMV sponge,

7 sites

ACTAGTCGAGTCAACATCAGGACATAAGCTAaaaTCAACATCAGGACATAAGCTAaaaTCAACATCAGGACATAAGCTAaaaTCAACATCAGGACATAAGCTAaaaTCAACATCAGGACATAAGCTAaaa TCAACATCAGGACATAAGCTA

CTAAAATCAACATCAGGACATAAGCTAAAATCAACATCAGGACATAAGCTA

F1:  AAACTCGAGTCAACATCAGGACATAAGCTAAAATCAACATCAGGACATAAGCTAAAATC    Xho I

F2:  (AAGCTAAAATCAACATCAGGACATAAGCTAAAATCAACATCAGGACATAAGCTAAAATCAAC)

TTCGATTTTAGTTGTAGTCCTGTATTCGATTTTAGTTGTAGTCCTGTATTCGATTTTAGTTG

F3:  TCT TTCGAA ATCGAATACAGGACTACAACTAAAATCGAATACAGGACTACAACTAAAAT   Hind III

R3:  TGTAAGCTTTAGCTTATGTCCTGATGTTGATTTTAGCTTATGTCCTGATGTTGATTTTAG

pSB-F1:  AAAACTAGTCGAGTCAACATCAGGACATAAG

actagt

pSB-R3:  TGTGACGTCGCTTTAGCTTATGTCCTGATGTT

g^acgt c

Sponge 5’-UCAACAUCAGGACAUAAGCUAaaaUCAACAUCAGGACAUAAGCUAaaaUCAACAUCAGGACAUAAGCUAaaaUCAACAUCAGGACAUAAGCUAaaaUCAACAUCAGGACAUAAGCUAaaa UCAACAUCAGGACAUAAGCUA-3’

                                AGAC

![](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e2096c319421a02dab28/1509417482595/)![](https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7e2096c319421a02dab28/1509417482595/?format=500w)

miR-21        3’ - AGUUGUAGUC    UAUUCGAU – 5’

Sponge    5’-…AAAUCA ACAUC AG    AUAAGCUA AAA…-3’

                                 GAC

（不是每次都1:3，要看每个盒子的说明书）

（It is not 1:3 for each time, which should refers the specification on each box）

溶胶1:3比例（加多了也没事）

Add the sol with the ratio of 1:3 (It doesn’t matter if the sol is added excessively)

琼脂凝胶DNA回收        1:3比例

Agar gel DNA recovery with a ratio of 1:3

1)     配方①0.96×3=2.88ml(21p)  2ml+880µ

1)         Formula ①0.96×3=2.88ml(21p)  2ml+880µ

②0.96×3=2.073ml  21s  2ml+73µ

 (这个枪最大容量是100，其实是1ml)

Maximum gun capacity is 100, 1ml in fact

2)         放入水浴锅37℃大约10分钟，中途要拿出来摇

2)         Put it into water bath at the temperature of 37℃ for 10 minutes, during which, it needs to be taken out for shaking

3）       放入吸附柱管里离心（可能需要离心多一次）

3）       Put it into the adsorption column tube for  centrifugation  which may need to be conducted for more than once

1min                                 1min:12000rmp          直接按start

1min                                 1min: 12000rmp         Directly press start

4）       把下面废液管里溶液倒掉

4）       Outwell the solution in the waste teflon tube under the adsorption column tube

5）       加入600µ漂洗液（W2）到吸附柱管里并且离心30秒。1min：12000转

5）       Add 600µwash liquid (W2) into the column tube and conduct centrifugation for 30 seconds at the rotate speed of 1min:12000rmp

6）       重复5）但是只加入500µ漂洗液W2

6)         Repeat step 5) except for adding only 500µwash liquid W2

7）       不加入任何溶液，空甩2min

7)         Conduct centrifugation for 2min without adding any solution

8)         晾干10min

8)         Dry it in the air for 10min

9)         把50µl（洗脱液）（TE）加入吸附柱管中，（关盖）静置1min→离心1min

9)         Add 50µl (eluant) (TE) into the adsorption column tube, (cover the lid) still standing for 1min →centrifugation for 1min

原理：260=1时            每µ=50 稀释100

Principle: when 260=1     each µ=50          dilute 100 times

50×100=5000

核酸 分离①保留一纸节构

Nucleic acid separation ① preserve a paper structure

②

紫外分光，确定浓度和纯度

Conduct ultraviolet spectrometry, and confirm the concentration and purity

DNA测序          核酸分纸分离（电泳）

DNA sequencing Nucleic acid molecules separation (electrophoresis)

核酸性质

Nucleic acid properties

核酸电荷           凝胶（电泳）    电场强度           电场方面           电泳环境

Nucleic acid charge          Eel (electrophoresis)           Electric field intensity     Electric field       Electrophoresis environment

RNA酶抑制剂

RNA sin Inhibitor

RNA纯度分析              DNA>1.8          RNA<2.0

RNA purity analysis        DNA>1.8           RNA<2.0

导入DNA的方法

The method of importing DNA

转化 微注射技术

Conversion micro-injection technique

转染

Transfection

克隆筛选

Clone selecting

实验：

Experiment:

配电泳用的胶1:2

The gel for preparing electrophoresis with a ration of 1:2

质粒距胶           1.0129ð2.024

Plasmid gel leaking         1.0129ð2.024

2017. 7.14

因为片段较短，所以不需要模板。

-引物的量和模板的有无根据亲本的长短而变

PCR 10ml：

-Taq酶 0.1μ

-Taq buffer 1 μ

-水：补足

-F.R primer ：每个0.3μ

-模板&浓度

-dNTP 0.9 μ

FR primer：10μ M

酶切

酶+buffer+片段（p\*）+水

37℃，4h， 片段双切；plasmid单切

\*PCR出来的意思？

连接：

-连接酶

-双酶切后质粒（A）

-双酶切后片段（B）    A：B= 1:3~1：6

16℃ 4h

转化--》挑单克隆--》摇菌--》质粒测序

上游>=200bp， 测800-1000bp

功能检查

E、F菌落成长失败，需要重新做PCR

PCR

-50μ 35组

-100μ （引物40个）

需要：

dNTP

4μ

×2

buffer

5μ

Forward f-20

20μ

r

20μ

Taq

0.5μ

电泳

配方：1. 琼脂糖凝胶 30ml ： 0.3g 琼脂糖 比例1：100

Goldview 1.8μ loading buffer、甘油、溴酚蓝

放入微波炉热到沸腾，沸腾3次后，再回归常温时， 加入Goldview （GV）形成凝胶后，放入电泳仪

1.    10μloading buffer， 加入50μ的PCR产物， 20μ~（marker？）

算法： 1:6=loading buffer：总溶液

2.  离心（随意）

3. 按启动两次， 开始跑胶 （25min），蓝色标记到胶的一半停

4. 带很清晰。放入凝胶成像分析仪并拍照

5. 在紫外灯下，能清晰看见（如果肉眼看不清的话），然后进行切胶回收

6. 先对空离心管进行称重 【1. 2016（+-0.001），1.984（21s）】

7. 切胶

8. 对离心管和胶进行称重，并计算胶的重量。

-2.975-2.015=0.96 （21p）

-2.675-1.984=0.691 （21s）

溶胶 1:3 比例 （加多了没事）

(不是每次都1：3，要看每个试剂盒的说明）

1》配方：

-0.96x3=2.88ml---》2ml+880μ

-0.96x3=2.073ml 21s  2ml+73μ

（注意枪的分度值和最小最大）

2》放入水浴锅 37℃ 大约 10分钟，中途还要拿出来摇

3》放入吸附柱管里离心（可能需要离心多一次）

1min     1min：12000rmp 直接按start

4》把下面废液管里溶液倒掉

5》加入600μ漂洗液到吸附柱管里， 离心30秒， 1min：12000rmp

6》重复5， 但是只加入500μ漂洗液W2

7》不加入任何溶液，空甩2min

8》晒干 10min

9》把50μ洗脱液（TE）加入吸附柱管中（关盖），静置1min-->离心1min

测浓度：

DNA吸光度 260.0mm  280.0mm 320mm

1) 灭菌纯水加入石英比色皿中，100μ校准

2) 模糊面对自己，一定不要摸光面。透光率100% 吸光度为0

3) 把皿里的水吸干净

4) 加入99μ水，样品1μ（21s），伸到里面，量少吹&混匀，得出的值x500=核酸浓度

5) 用21p重复做第四步

 21s 浓度：114.5  21p浓度： 265.000

琼脂凝胶 DNA回收 1:3比例

2017.7. 15

取昨天的结果

2017.7.16

P1 重选细菌 除RNA

P2 （含SDS）裂解E.coli   变性

P3 中和高盐分 DNA etc. 复性 上清中取质粒 有RNA酶

2017.7.17

RNA提取比DNA难， RNA易降解

PCR可用来鉴定          不同的方法：-增加可信度 -适应不同情况（目的）

免疫荧光

见到阴性克隆，提质粒，送测序

2017.7.19

链接产物全部加入到感受态 （摇菌 40-50 min， 使感受态恢复活性）

》看浓度转 50μ感受态+10μ产物

42℃ 92s 感受态    150 转 37℃  （180~200转比较合适）

》凃板：正面 37℃ 30min 后翻面

12h 后， 挑菌落 （不要超过16h， 会失去活性）

质粒只能转一个，具有不相容性

注意处理冷冻样品时， 扭住瓶口就行。手心不要碰滴管

Wuhan, China .   New York, USA

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