DNA Sequencing核酸定序設施

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  • 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.

實驗

 

 

 


  • 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。
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