DNA Sequencing核酸定序設施

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  • When can I recive the sequencing data?

 

I f your samples are send on Monday to Thursday before 10:00am, you'll recive the data on Bio201 system on the next work day before 9:00am. I f your samples are send on Friday before 10:00am then you'll recive the data on Bio201 system on the next Monday before 9:00am. I f there is any messages about stop working announced  by Central Personnel Administration, Executive Yuan,our core facility will be out of service,sequencing results will be delayed.


  • Can I use the 8-strip tubes?

 

If your samples are more than 8,you may use 8-strip tubes to hold the samples, empty well in the middle is not allowed. please lable the order #1 to #8 on the tube, requestition number and your name is required on the tape lable. If your samples are more than 48,you may use 96-well plate to hold the samples, empty well in the middle is not allowed. Please seal carefully, the order must  start from A01 to H01 for a row. Requestition number and your name is required on the tape lable. 


  • If I have question about my sequencing data, what kind of  the information that I should provide?

 

If you have the questions about sequencing results, please provide the following information while you contact us: (1)your name(2)requestation no.(3)date of submission(4)your question or your data so that we can quickly help you th solve the problem.


  • What should I do if some kind of the information is wrong on my sequencing application form?

 

If there is any information wrong on the request form, you may correct it by handwriting directly, after we recive it, we may correct thar on line for you. You may also contact us (2652-3924) to correct it on line directly .


  • How to cancel the sequencing application?

 

If you wold like to cancel the requestion, please dial 2652-3924,we may cancel it for you.
If you make a requestion then the samples are no show in 3 working days,we'll cancel your requestion automaticly . Our business hour is Monday to Friday am8:00 to pm 4:50.


  • If the printer is out of order in my lab, what should I do about my application form?

 

If the printer is out of order in your lab, no printing request form is generated, you may come to our lab in N719 of Biomedical science Institute with the samples, we could print it for you, and our business hour is Monday to Friday am8:00 to pm 4:50.  If you come to the core facility out of bussiness hour, you may leave an A4 size message into our mailbox, we'll help you to print out when we recive it. To protect the envirement, we suggest to use the recyled paper to print out the request form.


  • About the sequencing request form,  why the printing method and the way into the mailbox  need to be specified?

 


To print out the DNA sequencing request form, please use A4 paper unified , vertical print. into a mailbox way : the back -up paper , with the text of that side towards the front of the left ( there are marked above method mailbox ) . So we can come up with all the neat application form, in the same direction . If your application through a single curl , or fold , we are very difficult to sort out these requisitions . In addition, the laboratory staff will comply with your request form to plan the experiment, and will be bound the request forms into a book , if you do not comply,do the horizontal print , or narrow print , paper size is wrong, we will be very troubled , so please to comply with our rules .


  • Can more than 2 people use the same personal account in one Lab?

 

Yes, you may.But everyone who can use this account will read and download the data you should notice that. Also when you make a requestion, please mark your name on the requestion form in order we have to contact you. We strongly suggest that each one create your own account. 


  • What should I notice about filling personal information when I register in Bio 201 system?

 

Please filling with your real name (no nick name ),telephone no. of Lab (no cell phone no.),e-mail better be sicica.edu or shcool mail or @gmail.com. After uploading the sequencing data, we'll inform you via e-mail.


  • How to work out with a template containing prohibitive secondary structure?

 

Just notify us on DNA sequencing-work request form. We have proprietary protocols for those template with GC rich and secondary structure.


  • What notes should I take of preparing PCR product for template?

 

  1. Check the specificity of product by gel electrophoresis.
  2. Remove dNTP and primers by purification before sequencing.
  3. Add only one strand of primer.
  4. Minimize the time of UV exposure when processing gel extraction.
  5. Decrease the volume of eluate when processing gel extraction.

  • How do I determine the concentration of plasmid DNA?

 

  1. Check the DNA and RNA contamination by gel electrophoresis.
  2. Estimate the DNA concentration with a marker of known concentration.
  3. Determine the ratio of absorbance at 260nm and 280nm with a spectrophotometer

Unappropriated amount of DNA may cause sequencing to fail. Therefor, we have a guideline in page "Sample preparation".


  • What notes should I take of primer design?

 

  1. Length of primer should be about 18-24 bases。
  2. Tm of primer should be above 45℃. However, the annealing temperature in our protocol is 50℃.
  3. Minimize the self-dimerization and prevent secondary structures in primer.
  4. Check the specificity of primer binding site.

  • Why did I get a failed DNA sequencing?

 

Causes of failed DNA sequencing reactions:

 

  1. Poor quality DNA
  2. Degraded or failed synthesis primer
  3. Too much template DNA
  4. Wrong primer used
  5. Bad water
  6. Template DNA is contaminated by inhibitors (eg. salt, phenol, EDTA, RNA, protein…)
  7. Loss of the reaction during clean up
  8. Dead sequencing chemistry
  9. BigDye chemistry is stored under the wrong conditions or is freeze-thawed too many times. Either the Taq DNA polymerase or dye labeled nucleotides can have degraded
  10. Blocked capillary

 

 

​Solving DNA sequencing reaction failures:

 

  1. Poor quality DNA:
    Please using the Spectrophotometer to check DNA purity, make sure the extraction or Purification process is not contaminated by some inhibitors eg, salt, phenol, EDTA,RNA and protein, because those material will kill the sequencing reaction. Even you elute the DNA with incorrect water ( eg. Aicd pH, water with salt, water is contaminated with other nucleic acid, nuclease...) it will affect the sequencing results.
  2. Degraded or failed synthesis primer:
    Don't use old diluted primer stocks. Don't use other peoples stocks. If you have any doubt about how the primer quality through it out and make up a fresh working solution from the primer stock. If you suspect that the primer is poor quality either have it presbyters or check in a polymerase chain reaction (PCR). Alternatively if you have a control template that you know should work with the primer then this can be a good way of identifying primer problems.
  3. Too much template DNA:
    This can be avoid by checking the concentration of the template on an agarose gel before sequencing. This will also allow you to see the purity of the template DNA and if there is a significant amount of contaminating genomic DNA or RNA present. Do not rely on a spectrophotometer reading to calculate the template concentration.
  4. Wrong primer used:
    Check the sequence of the primer and template to make sure that the primer binding site is present. This can be a particular problem with some "universal" primer sequences which do not work with some common plasmids. Do not trust other people's working stock solutions and make your own. It might take 5 minutes longer, but it will save you a lot of future headaches.
  5. Bad water:
    Inhibitors (low Ph water, nuclease...) can end up in lab water stocks that can kill DNA sequencing reactions. If you think this may be a problem then throw out the water and use a fresh stock - remember water is cheap.
  6. DNA template is contaminated with inhibitor ( eg.salt, phenol, EDTA, RNA, protein…):
    Make sure the extraction or purification process is not contaminated by some inhibitor eg, salt, phenol, EDTA,RNA and protein, because those material will kill the sequencing reaction. Even you elute the DNA with incorrect water ( eg. Aicd pH, water with salt, water is contaminated with other nucleic acid, nuclease...) it will affect the sequencing results.
  7. Loss of the reaction during clean up:
    After sequencing reaction, we'll perform the ethanol precipitation protocol to clean up the sequencing reaction, There are a number of kits that work very well, unfortunately they can be very expensive. One tip for avoiding loss of the reaction DNA pellet when using the ethanol protocol is to add 1?l of a 20 mg/ml solution of glycogen (Sigma G-1508) to the sequencing reaction before adding the ethanol. This helps make the pellet visible and the glycogen does not seem to interfere with the injection of the sequencing fragments onto the sequencers capillaries.
  8. Dead sequencing chemistry:
    This is a relatively rare problem, however, if a batch of BigDye chemistry has not been used for some time, or there is any doubt about how it has been stored, then it is advisable to perform a control sequencing reaction before undertaking a large number of experimental reactions. Many problems with dead chemistry can be prevented by storing the BigDye chemistry in small aliquots and avoiding repeated freeze/thaw cycles.
  9. Blocked capillary:
    We'll clean our capillary systems every week in our lab, remove the bubbles as possible, we also change the filter periodically to avoid capillaries to be blocked.

  • Why does the newly prepared primer work but fail later?

 

  1. Low pH condition and contaminated with DNase may cause primer degradation.
  2. Mixing after thawing was insufficient for precise concentration of primer.
  3. Primer with repeated freezing thawing may be damaged.

  • Why I did not get the result of sequence on Bio201?

 

The sequence results have yet to upload if it shows “Empty” in Bio201. Moreover, you can consult any question with us on this number: 2652-3924.


  • What should I do if I cannot download sequence result or review the sequence file?

 

If the file is not corrupted after we check, we can mail you the sequence result by e-mail.


  • What software can I use to review sequence file except Bio201?

 

We recommend user to review sequence result with Sequence Scanner Software. It is available in Link page, or you can download it from here.


  • How long does it keep sequence result on Bio201?

 

We are not in charge of preservation of result data, please keep it safe indeed. All of data on Bio201 can only be retrieved for the past 6 months.

常見問答

送樣及訂單
 

 

 

 

 

 

 

 

 

 

​實驗

 

 

 

 

 

檔案存取

 

 

 


  • 請問何時可以收到定序結果?

 

周一到周四早上10點前送達之樣品一律在下一個工作日早上9點之前收到定序結果,週五早上10點之前送達之樣品將於下周一早上9點之前收到定序結果。如遇人事行政局宣布之停班或國定假日,本單位也會停止服務,前一日送達之定序服務將順延收到結果。


  • 請問多少樣品以上可以使用八連排管子,送樣方式為何?

 

當您樣品8個以上您就可以使用八連排管子送樣,必須8個為一排,中間不可以有空格或任意剪開,如最後不滿8的倍數請自行將最後的空管剪掉,必須在管子上標示您的順序告知#1和#8的方向,如有24個樣品請標示#1#8#9#16#17#24的位置即可,想要詳細標示1-24的也可以,最後請貼標籤標示清楚您的訂單編號和姓名,即可放置在冰箱內八連排專屬的架子上。如果樣品接近96個,歡迎使用96孔盤,順序請由A1~H1 8個為單位放置好,並用封膜封好,中間也請勿有任何空格,最後請貼標籤標示清楚您的訂單編號和姓名,直接放置在冰箱內。


  • 有任何定序上的問題打電話到定序實驗室需要告知那些資料?

 

很多人打電話來問問題都只報上您是哪家實驗室或是哪位老師家的助理,其實我們需要知道的是您的(1)姓名(2)訂單編號(3)送件日期(4)您的問題。樣品很多,請告知上述四大要件我們才能快速幫您解決問題。謝謝配合。


  • 請問訂單上樣品名稱或是樣品種類等資訊填錯了該怎麼辦?

 

可以直接在紙本訂單上面做修改,我們收到訂單後可以為您作申請變更。也可以撥電話2652-3924請定序實驗室人員幫您做修正。如整張訂單都填錯,建議您來電做取消,重新申請。


  • 請問該如何取消訂單?

 

如果重複申請相同的訂單或是臨時想取消訂單,請撥2652-3924告知定序實驗室幫您取消即可。如未告知取消時,訂單會一直存留在我們的申請系統內,三個工作天未送件,我們將自動幫您做取消。


  • 實驗室印表機壞掉了,定序申請單印不出來該怎麼辦?

 

早上8點到下午5點鐘的工作時間您可以親自送樣品過來時,直接到N719實驗室內找尋核酸定序的工作人員幫您列印即可。如果是下班時間,您也可暫時投入一張A4紙張於定序信箱內,留言請我們幫您列印。歡迎大家使用單面廢紙列印。


  • 核酸定序申請單的列印方式和投入信箱的方式為甚麼一定要規定格式呢?

 

請各位統一採用A4紙張,直式列印,投入信箱的模式是,反面朝上,正面的最前端朝左邊(信箱的上面有標示方法)。如此我們可以很工整的拿出來全部的申請單,不需要轉向。若您的申請單捲曲過,或是對折投入,我們都很難整理這些申請單。此外,實驗室的人員會遵照您的申請單做實驗的規劃,會裝訂成一整本,如果您不遵照規定印成橫的,或是縮小列印、紙張規格不對,我們在做實驗的時候,都會很困擾,所以麻煩各位要遵守我們的規定。


  • 同實驗室可以共用一個Bio201帳號來使用嗎?

 

如貴實驗室不在乎自己的定序結果可以供同實驗室同仁下載,貴實驗室可以使用同一個帳號申請定序,不過如遇上定序樣品置放等等有問題時,本實驗室會不知道該找哪一位同仁? 此時建議您在備註欄位上寫上送樣人的名字。


  • 新註冊Bio201帳號密碼時,填寫個人資料需要注意什麼事情?

 

請填寫中文真實姓名,請留實驗室電話,E-mail的部分請確定不會擋信,因為上傳結果後,系統會藉由e-mail通知您可以進系統看結果。


  • 如果序列有特殊結構該如何解決?

 

  1. 當您的DNA 序列出現 poly A 或 poly T,可在備註欄寫上有 Homopolymeric region。如定序結果不佳,建議您更換另一股的引子。
  2. 當您的 DNA 序列出現 GT、GA、AT、CT 等 repeat sequence 或 GC rich 的序列,請在備註註明您的樣品有 repeat sequence / GC rich,我們會作特殊處理。

  • PCR 產物定序要注意那些事項?

 

  1. 進行洋菜膠電泳時需呈現single band。
  2. 定序前要先純化去除 primer 和 dNTP。
  3. 只能加一股的 primer。
  4. 進行膠體萃取時,避免UV照射過久。
  5. 建議純化後回溶的體積少於protocol的一半,避免濃度太低。

  • Plasmid DNA 如何定量?

 

  1. 請先以洋菜膠電泳確認沒有 genomic DNA 或是 RNA 污染。
  2. 以定量的 marker 做概略的濃度估算
  3. 以 spectrophotometer 再次定量,雖然 A260的測定也會反映出其他種類的核酸如 genomic DNA 或是 RNA。

當 DNA 過少或是過多都會造成定序失敗,因此我們會有建議的濃度值範圍給各位參考。


  • 引子設計要注意什麼?

 

  1. 長度請介於18~28 bases.。
  2. Tm 值需大於45℃。本實驗室的annealing temp. 為50℃。
  3. 避免自黏或形成二級構造。
  4. 以軟體或網站預測引子黏合位置,避免存在多個引子黏合位。

  • 定序失敗的原因?

 

反應完全失敗(無訊號)

 

  1. DNA模板品質不好,請檢查:DNA的鹽類濃度過高、蛋白質殘留、有機溶劑殘留、本身降解或直接送菌液做定序。
  2. 引子方面:未加或加錯引子、本身或載體DNA降解以及序列設計錯誤。

反應不完全(微弱訊號)

 

  1. DNA模板品質不好,請檢查:DNA的鹽類濃度過高、蛋白質殘留、有機溶劑殘留、本身降解、濃度過低或被UV照射過。
  2. 引子方面:本身或載體DNA降解、序列設計錯誤、Tm值過低、引子互相鍵結或形成二級結構、濃度過多。
  3. 遇到特殊構造如GC rich、poly A / T、短片段重複序列。
  4. 受其他樣品干擾。

反應正常,但出現污染

 

  1. DNA模板方面:純化不全殘留dNTP及引子、RNA汙染、染色體DNA汙染、存有不同克隆的質體、多重PCR產物
  2. 引子方面:存有一種以上的引子、序列設計錯誤、Tm值過低、本身降解、模板具有多重引子黏合位
  3. 遇到特殊構造如GC rich、poly A / T、短片段重複序列。
  4. 受其他樣品干擾。
  5. 模板量過多。
  6. 存在其他螢光汙染物,如loading dye。

  • 為什麼剛溶解的引子可正常使用,過一段時間後卻無法使用?

 

  1. pH過低、遭菌或核酸水解酵素污染的回溶用水會使引子降解。
  2. 引子解凍後未混合均勻,造成取用量不準確。
  3. 重複的解凍、冷凍過程也會造成引子降解。建議將引子分裝成少量多管,減少重複解凍次數。

  • 為什麼我在Bio201系統上面看不到我的定序結果?

 

如結果顯示Empty,表示結果尚未上傳。如已經過了每日早上9點該上傳的時間仍顯示Empty有可能是因為當日件數太多,定序結果尚未能如期上傳,有任何的問題都請撥2652-3924來電詢問。


  • 如果無法下載檔案或是下載吃後無法開啟檔案該怎麼辦?

 

請與我們聯絡,如果我們可以下載也可以開啟檔案,將可以把下載的檔案以e-mail的方式寄給您。


  • 如果不在Bio201系統上觀看定序結果,可以下載甚麼軟體?

 

我們建議使用Sequence Scanner Software來觀看圖形檔,請到本網頁常用連結處點選下載或是從這裡下載。


  • 請問Bio201上面的定序結果保存多久?

 

基本上定序實驗室將結果上傳之後並不負責幫您保管結果,請您每次務必存檔,系統上的結果僅保存6個月。

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