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燈號說明

審定:無
翻譯:柯佳惠(簡介並寄信)
編輯:朱學(簡介並寄信)


實驗室介紹課程注意事項 (PDF - 1.7 MB)
Lab Introduction Lecture Notes (PDF - 1.7 MB)


實驗室計畫第一部份
Laboratory Project Part I

第一天 (第三堂課)
Day 1 (Lecture #3):
將4株帶有質體且已隔夜培養的大腸桿菌XL-1 blue (生長16小時) 進行接種。
Inoculation of 4 overnight cultures (16hr growth) of plasmid-bearing E. coli XL-1 blue.

第二天 (第四堂課)
Day 2 (Lecture #4):
利用離心來收集細菌,並將集中於底部的細菌細胞貯於-80℃。
Harvesting of the bacteria by centrifugation and storage of the cell pellets at -80°C

第三天 (第五堂課)
Day 3 (Lecture #5):
DNA製備 (有四個樣品)
DNA preparation (4 samples)
260 nm吸光值測定以定出DNA濃度
Absorption measurement at 260nm to determine the DNA concentration
準備DNA自動定序的樣品
Sample preparation for automated DNA sequencing

第四天 (在第六堂課的前一天)
Day 4 (a day before Lecture #6):
將樣品送去作DNA定序
Sample submission for DNA sequencing

第五天 (第七堂課)
Day 5 (Lecture #7):
DNA限制酶切割反應
DNA restriction digest
瓊脂洋菜膠體電泳
Agarose gel electrophoresis
定序結果
Hand-out of the sequencing results

你的工作:
Your Job:

  • 盡量找出所有與你的複殖株有關的資訊。
    Find as much information as possible about your clones.
    請記住複殖株是由動物模式生物而來的。為什麼雞是適合用來找尋內耳基因的模式生物?以雞來當作模式生物的缺點為何?
    Remember that the clones stem from an animal model. Why is the chicken an appropriate model for inner ear gene discovery? What are disadvantages of the chicken model?

    挑出一或兩個可能帶有對於內耳功能具有重要性的基因之複殖株。目前對這些基因已經知道那些事情?
    Pick one or two clones that may have significance for inner ear function. What is known about these genes?

  • 請從第一天開始就要盡你所能地詳細記載實驗記錄。在第16堂課或之前必須交出一份包含結果討論的實驗報告。
    Document your experiments as detailed as possible starting with day1. A written lab report including a discussion of the results has to be submitted on or before Lecture #16.

製備質體DNA─Qiagen方法
Plasmid DNA Preparation - Qiagen Method

也可見 www.qiagen.com - Qiaprep小量製備套組手冊
See also www.qiagen.com - QIAprep Miniprep Handbook

  1. 將沈澱的細菌細胞以 250µl 的緩衝溶液P1重新懸浮,並換到微量離心管。
    Resuspend bacterial cell pellet in 250µl buffer P1 and transfer to a microcentrifuge tube.
  2. 請先準備好計時器-下一個靜置步驟必須精確的在4~5分鐘完成 !
    Have a timer ready - the next incubation step has to be exactly 4-5min!
  3. 加入 250µl 的緩衝溶液P2,蓋上蓋子並且立即以上下翻轉5~10次的方式混合完全。
    Add 250µl buffer P2, close the tube and immediately mix by 5-10 inversions.
  4. 在剛好4分鐘時,加入 350µl 的緩衝溶液N3,蓋上蓋子並且立即以上下翻轉5~10次的方式混合完全。
    After exactly 4min, add 350µl buffer N3, close the tube and immediately mix by 5-10 inversions.
  5. 用桌上型微量離心機以最高速離心10分鐘。白色沈澱物含有蛋白質與細菌基因組DNA。
    Centrifuge for 10min at full speed in a tabletop microcentrifuge. The whitish pellet contains proteins and the bacterial genomic DNA.
  6. 將上清液換至 (Qiaprep) 離心管柱。請不要吸到任何來自白色沈澱物的東西。
    Transfer the supernatant into a spin (QIAprep) column. Do not transfer any material from the white pellet.
  7. 開啟真空裝置使液體通過管柱 (質體DNA會與管柱物質結合)。
    Switch on the vacuum to draw the solution through the column (The plasmid DNA binds to the column material).
  8. 加入 750µl 的緩衝溶液PE來洗滌管柱。
    Wash the column by adding 750 µl buffer PE.
  9. 將管柱換至微量離心管,並且於最高速離心一分鐘使管柱物質乾燥 (此步可移除殘留的酒精。酒精是緩衝溶液PE的成份之一。酒精會干擾之後的DNA操作)。
    Transfer the column into a microcentrifuge tube and spin for 1min at full speed to dry the column material. (This removes any ethanol, which is part of buffer PE. Ethanol will interfere with subsequent manipulations of the DNA).
  10. 將管柱置於一個乾淨的微量離心管。將離心管標上記號。加入 70 µl 的洗提溶液 (EB) 於管柱物質的中心 (但是不要碰到白色管柱物質) 來洗提出DNA。靜置一分鐘,接著於最高速離心一分鐘。
    Place the column in a clean microcentrifuge tube. Label the tube. Elute DNA by adding 70 µl of elution buffer (EB) into the center of the column material (do not touch the white column material though). Let sit for 1min and then centrifuge at full speed for 1min.

質體DNA濃度的測定:
Determination of the Plasmid DNA Concentration:

  1. 將你的每個質體DNA的各取 5µl 來稀釋20倍:這是1:20的稀釋,所以代表你要取 95µl 經MilliQ過濾的無菌水與 5µl 的質體DNA溶液混合。
    Dilute 5µl of each of your plasmid DNA 20-fold: This is a 1:20 dilution and means that you mix 95µl sterile MilliQ-filtered water with 5µl plasmid DNA solution.
  2. 你的空白組為經MilliQ過濾的無菌水。你使用的波長為260nm。
    Your blank will be sterile MilliQ-filtered water. Your wavelength is 260nm.
  3. 以MilliQ水來矯正紫外線分光光度計。小心!石英管又小又貴,且掉地就會破!
    Blank the UV-spectrophotometer with MilliQ water. Careful, the Quartz cuvette is small, expensive, and it breaks when dropped on the floor!
  4. 將水換成你的已稀釋樣品並且讀取樣品的數值。
    Exchange the water with your diluted sample and read sample.
  5. 在測定DNA液體時,每一A260單位有 50µg DNA/ml的濃度。
    1 A260 unit of a DNA solution has a concentration of 50µg DNA / ml.
  6. DNA濃度以 µg/ml 表示 = 讀取的A260值 × 稀釋倍數 × 50
    DNA concentration in µg/ml = A260 reading * dilution * 50
  7. 請記得:500 µg/ml 等於 500 ng/µl。
    Remember, 500 µg/ml equals 500 ng/µl

準備定序用的樣品
Sample Preparation for Sequencing:

  1. 定序人員需要 10µl 濃度為 200 ng/µl的DNA。換句話說,他們需要 2µ的質體DNA。
    The facility asks for 10µl DNA solution at a concentration of 200 ng/µl. In other words, they want 2µg of plasmid DNA.

    如果你的DNA濃度大於 200 ng/µl,你必須稀釋至適當的量。
    If your DNA concentration is above 200 ng/µl, you need to dilute the appropriate amount.
    如果你的DNA濃度小於 200 ng/µl 但大於 100 ng/µl,請給他們 10µl 的DNA溶液 (因此你至少給他們1μg)。
    If your DNA concentration is below 200 ng/µl but above 100 ng/µl, give them 10µl of your DNA solution (therefore you give them at least 1µg).
    如果你的DNA濃度小於 100 ng/µl,你的DNA量太少以致無法成功地進行DNA定序。
    If your DNA concentration is below 100 ng/µl, your yield is too low for successful DNA sequencing.

  2. 將你的樣品編號標於離心管蓋上面,編號的後面接著寫破折號與T,T表示模板 (template) (例如:5A-T或3C-T)。
    Label the tubes on top with your sample number, followed by a dash and a T for template (for example 5A-T or 3C-T).
  3. 我們使用濃度為 10 ng/µl 的標準DNA定序引子─每個定序反應我們需要 10µl 的寡核苷酸溶液。此溶液會有提供。定序引子會黏合至pAD-Gal4的5’複殖位置 (EcoRI) 上游。
    PAD-Gal4前讀序列 5’ GGAATCACTACAGGGATG 3’
    We will use a standard DNA sequencing primer at a concentration of 10 ng/µl - We will need 10µl of this oligonucleotide solution per sequence reaction. This solution will be provided. The sequencing primer will anneal upstream of the 5' cloning site (the EcoRI) of pAD-Gal4.
    pAD-Gal4 forward seq 5' GGAATCACTACAGGGATG 3'


實驗室計畫第二部分
Laboratory Project Part II

第六天 (第7堂課):
Day 6 (Lecture #7):
DNA限制酶切割反應
DNA restriction digest
瓊脂洋菜膠體電泳
Agarose gel electrophoresis
定序結果
Hand-out of the sequencing results

你的工作:
Your Job:

  • A) 盡量找出所有與你的複殖株有關的資訊。
    A) Find as much information as possible about your clones.
    請記住複殖株是由動物模式生物而來的。為什麼雞是適合用來找尋內耳基因的模式生物?以雞來當作模式生物的缺點為何?
    Remember that the clones stem from an animal model. Why is the chicken an appropriate model for inner ear gene discovery? What are disadvantages of the chicken model?

    挑出一或兩個可能帶有對於內耳功能具有重要性的基因之複殖株。目前對這些基因已經知道那些事情?
    Pick one or two clones that may have significance for inner ear function. What is known about these genes?

  • B) 請從第一天開始就要盡你所能地詳細記載實驗記錄。在第16堂課或之前必須交出一份包含結果討論的實驗報告。
    B) Document your experiments as detailed as possible starting with day1.
    A written lab report including a discussion of the results has to be submitted on or before Lecture #16.

DNA限制酶切割反應
DNA Restriction Digest

每個質體含有一段cDNA,此插入基因是利用EcoRI與XhoI來複殖的。因此,以限制酶 EcoRI與XhoI來進行雙重切割反應後,應該會把插入基因從質體釋出。
Each plasmid contains a cDNA insert that was cloned via EcoRI and XhoI. Consequently, a double-digest with the restriction endonucleases EcoRI and XhoI should release the insert from the plasmid.

為了能在吸取液體時操作方便,我們會利用已經含有適當緩衝溶液與兩種限制酶的酵素反應混合液來操作。
To make pipetting easier, we will use an enzyme Master Mix that already contains the appropriate buffers and the two restriction enzymes.

  1. 將酵素反應混合液取 10µl 分裝至每管要被分析DNA至反應管。請將管子作上記號。
    Aliquot 10µl of the master mix for each DNA to be analyzed into a reaction tube. Label the tubes.
  2. 從每管DNA各取 10µl 的DNA液體加入酵素反應混合液中。
    Add 10µl of each DNA solution to the Master Mix.
  3. 稍為離心使DNA與酵素的混合液集中於管子底部。
    Collect the 20µl of DNA and enzyme mixture on the bottom of the tube by brief centrifugation.
  4. 置於 37°C 45~90分鐘。
    Incubate at 37°C for 45-90 min.
  5. 加入 7µl 的DNA 樣品追蹤染劑緩衝液,以膠體電泳分析並且與大小已知的DNA片段標記作比較。
    Add 7µl DNA loading buffer and analyze by Gel electrophoresis and comparison with Marker DNA fragments of known size.

Master Mix (2倍濃度):
Master Mix (2x concentrated):

280µl 無菌的MilliQ水
280µl sterile MilliQ water
80µl 10x 反應緩衝液 (New England Biolabs公司)
80µl 10x buffer (New England Biolabs)
20µl EcoRI 酵素 (10-20U/μl)
20µl EcoRI enzyme (10-20U/µl)
20µl XhoI 酵素 (10-20U/μl)
20µl XhoI enzyme (10-20U/µl)

這裡的master mix其實就是酵素所需要的buffer加上酵素再加上水,然後混成一大管,在實驗室如此操作的原因主要有吸取溶液較方便、加快實驗速度與實驗因素控制等原因。因為在實際上的實驗中,buffer與酵素的量並不需要太多,有時只要0.2 ul,連吸都不用吸,光是沾在管壁上的就已經大於0.2 ul,即使我們使用可以吸取0.2 ul的儀器,因為技術限制的緣故,不論準確與精確度都非常地大。例如我有六管樣品,每管需要0.2 ul酵素、1 ul buffer與8 ul的水,相較於六管分別慢慢加,不如一次吸取六管或7管的份量於一管內 (此時這管就是master mix),然後一次再吸取9.2 ul分裝到六管。如果有上百個樣品要操作,混成一大管再分裝也可加快實驗速度。”擴增反應液”不是不可以,如果現在的實驗是作PCR的話,我是會用這個名詞,但說穿了道理跟此處作限制酶切割是一樣的,而且PCR實驗要加的東西更多,實驗條件也更tricky,更需要master mix,所以有很多廠商在賣master mix給醫院,因為醫事檢驗需要操作大量的sample。

瓊脂洋菜膠體電泳:
Agarose Gel Electrophoresis:

  1. 請穿戴手套!因為電泳用的塑膠製品可能被用來觀察DNA的致癌物溴化乙錠所汙染。
    Wear gloves because the electrophoresis plasticware could be contaminated with the carcinogen Ethidiumbromide, that is used to visualize DNA.
  2. 將1克的瓊脂洋菜粉末加入100毫升的膠體電泳緩衝液並煮沸 (微波爐:45秒,晃幾下, 20秒, 晃幾下, 20秒)。等10分鐘待其降溫。
    Boil 1g Agarose in 100ml Gel running buffer (microwave: 45sec, swirl, 20sec, swirl, 20sec). Let cool for 10min.
  3. 加入 3µl 的溴化乙錠,並倒入組合好的膠體盤。
    Add 3µl of Ethidiumbromide and pour into assembled gel tray.
  4. 待膠體凝固 (15分鐘)。
    Let gel solidify (15min).
  5. 將DNA樣品注入 (每個凹槽 15-20µl)。
    Load DNA samples (15-20µl/well).
  6. 以100V進行電泳30~45分鐘,並以紫外光觀察DNA片段 (請配戴護眼罩!)。
    Run at 100V for 30-45min and visualize DNA fragments with UV light (wear eye protection!).
  7. 取得你的膠體影像印出圖,並與標記比較後定出插入基因的大小。
    Get a printout of your gel and determine the inserts' sizes by comparison with the markers.



 
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