- 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?
- Check the specificity of product by gel electrophoresis.
- Remove dNTP and primers by purification before sequencing.
- Add only one strand of primer.
- Minimize the time of UV exposure when processing gel extraction.
- Decrease the volume of eluate when processing gel extraction.
- How do I determine the concentration of plasmid DNA?
- Check the DNA and RNA contamination by gel electrophoresis.
- Estimate the DNA concentration with a marker of known concentration.
- 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?
- Length of primer should be about 18-24 bases。
- Tm of primer should be above 45℃. However, the annealing temperature in our protocol is 50℃.
- Minimize the self-dimerization and prevent secondary structures in primer.
- Check the specificity of primer binding site.
- Why did I get a failed DNA sequencing?
Causes of failed DNA sequencing reactions:
- Poor quality DNA
- Degraded or failed synthesis primer
- Too much template DNA
- Wrong primer used
- Bad water
- Template DNA is contaminated by inhibitors (eg. salt, phenol, EDTA, RNA, protein…)
- Loss of the reaction during clean up
- Dead sequencing chemistry
- 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
- Blocked capillary
Solving DNA sequencing reaction failures:
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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?
- Low pH condition and contaminated with DNase may cause primer degradation.
- Mixing after thawing was insufficient for precise concentration of primer.
- 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.
- 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。
- 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。
- 進行洋菜膠電泳時需呈現single band。
- 定序前要先純化去除 primer 和 dNTP。
- 只能加一股的 primer。
- 請先以洋菜膠電泳確認沒有 genomic DNA 或是 RNA 污染。
- 以定量的 marker 做概略的濃度估算。
- 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。
- 長度請介於18~28 bases.。
- Tm 值需大於45℃。本實驗室的annealing temp. 為50℃。
- 遇到特殊構造如GC rich、poly A / T、短片段重複序列。
- 遇到特殊構造如GC rich、poly A / T、短片段重複序列。
- 存在其他螢光汙染物，如loading dye。
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。