The MCIC provides services for
- low throughput sequencing using the ABI 3100xl and the 3'-BigDye dideoxynucleotide triphosphates labeling chemistry,
- genotyping using various platforms: fragment analysis on Beckman CEQ8800, SNP analysis on Luminex200 or Illumina BeadXpress
- MPS sequencing using the Illumina Solexa Genome analyzer.
Sequencing
Instructions for sample submission
The ABI Prism 3100xl genetic analyzer is a 16-capillary instrument using fluorescence based electrophoresis technology for DNA sequencing and fragment analysis. For sequencing we routinely use the 3'-BigDye dideoxynucleotide triphosphates labeling chemistry.
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Sample submission in microtiter plates:
If you have 8 or more reactions, submit samples in low profile thin-walled, V-shaped microtiter plates (see for example image; Fisher catalogue #05-500-63) or strips. Make sure that the sample order of the microtiter plate or strip follows the sample order in your "Sequencing Sample Form". Label samples following the columns. Bring the following quantities:
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| List samples following the columns | |
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| Low profile thin-walled, V-shaped microtiter plate |
Submission of templates to be sequenced with custom primers:
Pre-mix template and primer as follows: 4ul of DNA (see concentrations in the table below) and 2 ul of custom primer at 2 pmoles/ul (or app. 5ng/ul) in H2O.Submission of templates to be sequenced with primers available at the center:
Submit 4 ul of template DNA in H2O or 10mM TrisCl pH8.0 buffer (no EDTA!) at the concentrations indicated in the table below.
Submission of single reactions
Use this method only if you have less then 8 samples. Bring the samples in microcentrifuge/eppendorf tubes. Use black Sharpie markers to label your tubes. Limit sample name to up to 5 characters (the simpler the name, the less likely we will confuse the samples). Do not overlap tape, so that the tubes can still fit in the microcentrifuge. Note that when you submit samples in individual tubes, we ask for a slightly larger volume because we need to transfer them into microtiter plates.
Submission of templates to be sequenced with custom primers
Pre-mix template and primer as follows: 5ul of DNA (see for concentration above) and 2.5 ul of custom primer at 2 pmoles/ul (or app. 5ng/ul) in H2O.Submission of templates to be sequenced with primers available at the center
Submit 5ul of template DNA in H2O or 10mM TrisCl pH8.0 buffer (no EDTA!) at the concentrations indicated in the table below.
| Amounts of DNA needed: |
| PCR products | app. 2 ng/100bp/ul |
| ssDNA | 20 ng/ul |
| Plasmid | 100-150 ng/ul * |
| Phage, cosmid, BAC DNA | 500 ng/ul |
| Bacterial genomics DNA | 1ug/ul |
| Primers available at the center: |
| M13F | 5'-CGC-CAG-GGT-TTT-CCC-AGT-CAC-GAC-3' |
| M13R | 5'-AGC-GGA-TAA-CAA-TTT-CAC-ACA-GGA-3' |
| T3 | 5'-ATT-AAC-CCT-CAC-TAA-AGG-G-3' |
| T7 | 5'-TAA-TAC-GAC-TCA-CTA-TAG-GG-3' |
| SP6 | 5'-ATT-TAG-GTG-ACA-CTA-TAG-3' |
Tips for sequencing sample preparation
General recommendation for primer design
Your decisions concerning primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data. The purpose here is to provide you with some guidelines to follow in making those decisions.
- Primers should be at least 18-20 nucleotides in length to minimize secondary hybridizations.
- Primers with long runs of a single base should be generally avoided; it is especially important to avoid 3 or more G or C runs.
- Primers with melting temperature above 50 degree C generally produce better results than primers with lower melting temperatures.
- Primers should have G/C content between 50 and 60%.
- For primers with a G/C content of less than 50%, it may be necessary to extend the length beyond 18 bases.
- Primers should be “stickier” on their 5’ end than on their 3’ end. A “sticky” 3’ end, as indicated by a high G/C content, could potentially anneal and prime the polymerization reaction at multiple sites.
- A G or a C is desirable at the 3’ end of the primer.
- Primers should not contain complementary sequences (palindromes).
- Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself or to other primer molecules.
- Run a computer search against the vector and insert DNA sequences to verify that the primer and especially the 8-10 bases of its 3’ end are unique.
- Do not design degenerate primers, and do not request inosine for sequencing primers.
The quality of template DNA has a major impact on the quality of the sequence data. Inadequate template preparation is the most common cause of sequencing problems.
Methods for plasmid template purification:
QIAGEN DNA isolation (QIAGEN Inc., Chatsworth, CA; http://www1.qiagen.com/Products/DnaCleanup/PcrCleanup.aspx) and alkaline lysis with PEG precipitation (Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, p. 1.38 - 1.41 and p. 4.29 - 4.32) have proven to be the most reliable DNA preparation techniques for sequencing. These two methods generally provide good yields and clean DNA. Alkaline lysis followed by PEG precipitation is also inexpensive, the ingredients are readily available and the yields are generally higher that using other methods. Qiagen also offers single reaction kits or higher throughtput/96 well formats. There are many other methods and kits available on the market (5’ 3’ Perfect Prep; Promega Wizard Prep; Qiagen QiaWell and Tips; Nucleobond AX; .....). With these methods better results can be obtained by re-precipitating the final solution, rinsing the DNA pellet with 70% EtOH and resuspending the DNA in deionized water. The final DNA concentration and purity has to be accurately measured.
Host Strain variability in template preparation:
The host strain used for plasmid preparation also impacts template quality. The following information may help you in choosing a host strain: HB101 and DH5a host strains consistently produce good results; XL Blue, MV1190 and JM109 host strains show some variability in result quality; JM101 generally does not work well.
Cleaning a failed template preparation: It is sometimes possible to clean up a template which failed to give good sequencing data. Here are three commonly used methods:
- Extract DNA 2x with 1 volume of chloroform:isoamyl alcohol (24:1, vol:vol ratio). Precipitate the aqueous layer with EtOH and wash the DNA pellet with 70% EtOH.
- Precipitate with PEG. Add 0.16 volumes of 5M NaCl and 1 volume of 13% PEG 8000. Incubate 20 minutes on ice, centrifuge at 4oC for 20 minutes. Wash with 70% EtOH.
- Purify the DNA by ultrafiltration (for example, with Centricon-100, Micro-Concentrator columns, Perkin-Elmer Biosystems, Foster City, CA).
PCR products can be sequenced without cloning. After PCR amplification, the resulting PCR product exists in the presence of PCR primers, dNTPs, enzyme and buffer components. Dye terminator cycle sequencing requires rigorous PCR product purification, because the presence of residual primer or RNA will result in high background due to unspecific elongation products. Contamination with enzymes (nucleases, polymerases), salts, nucleotides, and ethanol decreases read length and signal intensity. PCR products can be purified directly from the PCR reaction using one of the following techniques, as long as only one product is present:
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Column purification, using commercially available kits, such as the QIAquick PCR purification columns, available for single reaction or 96-well format (QIAGEN Inc., Chatsworth, CA; http://www1.qiagen.com/Products/DnaCleanup/PcrCleanup.aspx), and Centricon-100 Micro-Concentrator columns (P/N N930-2119, Perkin-Elmer Biosystems, Foster City, CA).
- PCR products can be also purified by eluting them from the gel using electroelution, glass resin or other standard methods (QIAGEN or Promega kits are available).
- PEG precipitation is a very cost effective way for PCR fragment purification ("Fractionation of DNA Fragmebnts by Polyethylene Glycol Induced Precipitation" by John T. Lis, Methods in Ezymology, Vol.65, 1980. pp. 347 - 353). Get protocol.
- ExoSap (USB, cat.# 78200; http://www.usbweb.com/category.asp?cat=144&id=78200) is a mixture of Exonuclease I and Shrimp Alkaline Phosphatase, which respectively degrades the primers and hydrolysis the nucleotides used for the PCR reaction, and is added direcly to the PCR reaction. After the disegstion and inaclivation of the enzymes, the product is readty to sequence. This method is slighly mnore costly, but it requires very little manipulation and it produces excellent results.
If more than one PCR product is present, the desired product needs to be eluted from the gel, or the PCR reaction needs to be optimized to obtain a single product. PEG precipitation might work, as it is selectively precipitates DNA fragment of a certain sizes.
The chromatograms from the ABI 3100xl are saved as .ab1 or .scr files, and can be viewed with one of following software (click on the name to download the software):
- TraceViewer for Windows or Mac
- Xplorer for Windows or Mac
- Finch for Windows or Mac
- Chromas145 for Window/Window NT
- 4Peaks for Macintosh OsX and higher
- Sequence Scanner
How to retrieve your sequence data
You will be notified via e-mail, when your sequence data are ready to be downloaded from the OARDC server. Follow the link http://www.oardc.ohio-state.edu/dnaseq, or download your data using a file transfer program (FTP for PC users, Fetch for Mac users), with the following settings:
host: ftp.oardc.ohio-state.edu
user id : xxxxxxx
password: xxxxxx
User id and password are provided by the MCIC staff upon request.
You will receive 4 files: 1) chromatograph .ab1 format; 2) chromatograph .scf format; 3) phed confidence scores; 4) sequence fasta file. You are responsible to back up your data: the data will be deleted from the server after 30 days.
In house (OARDC appointment): $9.00per sample/for single reactions, or $6.00/sample for half (48 reactions) or full (96 reactions) microtiter plate (multiples of 48 reactions need to be submitted for the discount price.