FAQs for Using DNA Sequencing Service:
 
Frequently asked questions:
 
 
A.   What types of DNA template would not be sequenced if using the Big Dye for DNA sequencing reaction?
1.    Poly A, poly T, poly C, and poly G:
-       The template is a PCR product and has poly A and poly T bases > 10, then it would not be read through.
-       If poly A and poly T bases < 25 and is presented in plasmid DNA, then it would be sequenced.
  1. Templates with GT, GA, AT, CT, etc. repeats
  2. To sequence GC-rich templates, will need to have certain specific modifications to the standard reaction conditions. Such reaction will need to handle separately.
  3. Templates that are similar to GTGT, GGGGAGGA, GGGGTGGT, etc.
B.   What steps should users be aware of while performing the miniprep isolation of plasmid DNA for sequencing?
  1. After washing with solution but before eluting DNA from the column, be sure to centrifuge for an additional 3 to 5 minutes to remove residual ethanol.
  2. Please use heated water to elute DNA; the optimal water temperature is above 70°C.
  3. Residual alcohol, salts, EDTA, detergents, and organic chemicals may inhibit or interfere the Big Dye reaction.
C.   What steps should users be aware of while preparing PCR product for DNA sequencing?
  1. PCR product has to run as a single band on an agarose gel. A smeared result on the gel is not acceptable.
  2. No residual loading dye is allowed.
  3. PCR primers and dNTPs must be removed prior to sequencing.
  4. Only one set of new primer is allowed to add into the purified DNA template for sequencing.
  5. Avoid using short-wave UV light while extracting PCR product from agarose gels.
D.   How to use enzymes to purify PCR product?
  1. The Exonuclease I treatment degrades primers left over and the Shrimp Alkaline Phosphatase (SAP) degrades nucleotides left over after the PCR.
  2. PCR product has to run as a single band on an agarose gel.
E.   What is the recommended amount of template and primer to use for sequencing?

Type of Template
Amount
PCR Product* 200 – 500 bp
3 – 4 μl
PCR Product* 500 – 1000 bp
5 – 7 μl
Plasmid DNA < 15 kb, 60 – 100 ng / μl
8 μl
Primer
120 – 150 ng

*In the preparation of PCR products, be sure that the staining of the 2 μl PCR product band is stronger than the 500-bp band staining of the 2 μl 100 bp DNA Ladder Marker during the agarose gel electrophoresis. (PCR: Volume = 10 μl; in 30 cycles)
 
F.    What steps should users be aware of while designing a primer?
  1. Primers should be at least 18 bases in length.
  2. The Tm should be between 45°C and 55°C; annealing temperature is 50°C.
  1. Avoid primers that have secondary structure or that form self-dimers.
G.   Why samples/primer that worked in the PCR reaction was not working in the DNA sequencing?
It is because that the extension temperature of PCR reaction is 72°C and the extension temperature of DNA sequencing is 60°C. Therefore, for DNA sequencing, the Tm between 45°C and 55°C is preferred.
H.   What criterion is used to detect a weak reaction during the DNA sequencing?
To use the computer analyzed raw data as a standard, if the peak on the position 12000 at the x-axis does not reach 200 at the y-axis, then it shows a weak reaction. Special sequences are excluded in this case. In other words, the readable length of reaction is less than 550 bp.
I.      Causes of DNA sequencing failures:
  1. Failure of the DNA sequencing reaction:
(a) Poor quality of DNA template.
(i)            Salt concentration is too high
(ii)          Protein is present
(iii)         Residual organic chemicals are present, such as phenol, chloroform, ethanol, etc.
(iv)         Residual EDTA, detergents, etc. are present
(v)          Degraded DNA
(vi)         Bacterial liquid culture is used directly for sequencing
(b) Failure of the sequencing primer:
(i)            No primer is added
(ii)          Wrong primer is used
(iii)         Degraded primer
(iv)         Incorrect primer design
(v)          Primer binding site of vector is degraded
  1. Incomplete sequencing reaction:
(a) Poor quality of DNA template.
(i)            Salt concentration is too high
(ii)          Protein is present
(iii)         Residual organic chemicals are present, such as phenol, chloroform, ethanol, etc.
(iv)         Residual EDTA, detergents, etc. are present
(v)          Low DNA concentration
(vi)         Degraded DNA
(vii) Checked under the short-wave UV light
(b) Failure of the sequencing primer:
(i)   Incorrect primer design, like having 1 – 2 incorrect bases or the Tm is less than 45°C
(ii)          Degraded primer
(iii)         Primers that have secondary structure or that form self-dimers
(iv)         Too much primer (> 16 pmole) was used
(v)          Primer binding site of vector is degraded
(c) Encountered with special structured sequences, such as GC-rich, poly-A, T, CA, GA, CT, and GT repeats.
  1. The sequencing reaction is normal, but contamination is occurred:
(a) About DNA template:
(i)            Excess PCR primers and dNTPs residual from PCR
(ii)          RNA contamination
(iii)         Chromosomal DNA contamination
(iv)         Two different clones of plasmid are present
(v)          PCR product is not a single band on an agarose gel
(vi)         DNA template is too short
(b) Failure of the sequencing primer:
(i)            More than one primer is present
(ii)          Incorrect primer design, like having 1 – 2 incorrect bases or the Tm is less than 45°C
(iii)         More than one DNA binding site is present
(iv)         Poor quality of primer
(c) Encountered with special structured sequences, such as GC-rich, poly-A, T, CA, GA, CT, and GT repeats.
(d) The sequencing reaction is abnormal (too strong).
J.    How to open/read the sequencing data from a personal-use computer?
There are several softwares available online. Feel free to download to use.
  1. Chromas (for Windows) or Edit View (for Mac)
  2. FinchTV (for Mac OS X, Windows, Linux, and Solaris) at the Geospiza website
  3. Sequence scanner software from the website of the sequencing instrument company
K.   How to open/read the peak data of fragment analysis from a personal-use computer
The software, Peak scanner, can be downloaded from the website of the sequencing instrument company to use.
 

 

公告日:2011/07/13





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