 The Technology:The method of automated DNA Sequencing, utilizes the BigDye Terminator cycle sequencing chemistry from Applied Biosystems (ABI), ABI PRISM 3730xl DNA Analyzer and the ABI’s Data collection and Sequence Analysis softwares.
Download software:
For viewing and printing sample files containing the sequencing data as a electropherogram or text file
Sample submission:
When submitting your DNA samples, always enclose the following:
(type the details to prevent reading errors)
DNA samples and primers should be submitted only in tubes of 1.5 ml or full plates of 96 samples.
Write you name and name of DNA/primer on the tube's cap.
Sequence data is manually edited and sent to the customer in a .ab1 and .seq format via E-mail. Other options are available upon request. Please keep the name of your sample that is given by the software. For example;
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Run number
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Lane number
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two first characters of customer's name
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DNA ID
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Primer ID
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Name of file
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Type of file
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s1287_
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05_
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mi-
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pgem-
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T7
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.ab1.seq
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text
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s1287_
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05_
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mi-
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pgem-
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T7
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.ab1
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electropherogram
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DNA samples - 10µl per reaction:
Plasmid 75-300 ng/µl
PCR fragment 5-50 ng/µl
BAC or Cosmid 200-1000 ng/µl
DNA requirements:
All samples should be dissolved in double distilled water (DDW)
Spectrophotometric absorbance parameters of your sample should be:
260/280 = 1.8-2.0
260/230 = 1.8-2.0
Difficult Templates
If you are having trouble with difficult templates, such as those with high G+C content, homopolymer regions, secondary structures or siRNA and our standard reaction cannot solve the problem, we can run the reaction with 5% DMSO and Betaine. However, we cannot always sequence these regions effectively. Please mark the right column on the submission form
Host Strain
1. The host strain used for your plasmid can affect the sequence quality of the resulting DNA.
2. Hosts that consistently work well: HB101, DH5a.
3. Hosts that have demonstrated some variability: MV1190, XL1 Blue, JM109.
4. Grow more slowly than most strains, resulting in lower DNA yields.
5. Do not respond to TB growth medium, unlike other strains.
6. Hosts that do not work well: JM101.
If standard primers (for plasmid analysis) are to be used (see below), primer’s name will suffice. In more specialized analyses, please submit your own primers.
Internal primers submitted by the customer should be:
10µl per reaction, of 2 pmoles/µl (2µM), in DDW.
18-22 bases with 50-60% GC content. Melting point (Tm)= 50-600C.
Degenerate primers should be 50 pmoles/µl
Standard Primers available at the DNA sequencing facility:
Primer name Primer sequence
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T7 TAATACGACTCACTATAGGG
T3 AATTAACCCTCACTAAAGGG
M13-(-20) forward TGTAAAACGACGGCCAGT
M13-Reverse GGAAACAGCTATGACCATG
SP6-19mer GATTTAGGTGACACTATAG
SP6-20mer CCAAGCTATTTAGGTGACAC
SP6-18mer ATTTAGGTGACACTATAG
PolyT TTTTTTTTTTTTTTT /A /G /C
SK CGCTCTAGAACTAGTGGATC
KS TCGAGGTCGACGGTATC
pGEX 5': 5' - GGG CTG GCA AGC CAC GTT TGG TG - 3'
pGEX 3': 5' - CCG GGA GCT GCA TGT GTC AGA GG - 3'
T7term: 5' - GCT AGT TAT TGC TCA GCG G - 3'
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DNA Sequencing