DNA Sequencing of Plasmids and PCRs

The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.

  • Higher optical sensitivity and advanced polymers enable you to obtain higher-quality sequencing data for less.
  • Multiple automation features decrease costly human errors.
  • Optimized polymers increase your productivity without compromising your results.
  • Perform a wide variety of sequencing and fragment analysis applications including resequencing, microsatellite analysis, AFLP, LOH, SSCP, SNP screening and SNP validation.

 Note: To view your sequences, you can download free software from CLC or  FinchTV

Submitting Samples

Submission of a Plasmid for Primer Addition and Sequencing

  1. Please submit clearly labeled eppendorf tubes or V-bottom or PCR plates containing template DNA at ≥ 100ng/µl.  Do not submit samples in a flat-bottomed plate.
  2. A minimum of 7µl of template for each reaction is necessary but more is also acceptable.
  3. If you will be using a primer other than the universal primers that we provide, please submit at least 5µl per reaction of a clearly labeled 3.2pmol/µl stock.
  4. Be sure to include the sample name and primer that is to go with each sample on the online submission form.

Submission of PCR Products for Primer Addition and Sequencing

  1. Please submit clearly labeled tubes containing PCR products at a concentration of ≥50ng/µl.
  2. A minimum of 7µl of template for each reaction is necessary but more is also acceptable.
  3. If you will be using a primer other than the universal primers that we provide, please submit at least 5µl per reaction of a properly labeled 3.2pmol/µl stock.
  4. Be sure to include the sample name and primer that is to go with each sample on the submission form.

Submitting Template and Primer Already Combined for Sequencing

  1. Please use a maximum of 4µl volume containing template (plasmid=200-300ng total or PCR product=10ng/100bps of product) plus 1ul of 3.2pmol/ul primer per tube or well.
  2. Note that 500-600ng of DNA is suggested for templates around the size of pUC (~2.9kb). More DNA might be needed for larger templates.
  3. You can submit these samples frozen or dried down. If they are not dried down, please be sure to seal them properly so that evaporation does not occur.
  4. Fill out the on line Sequencing Submission Form.

Submission of PCR Products for Clean-up and Sequencing

  1. Please submit clearly labeled tubes containing PCR reactions at a minimum volume of 25µl.
  2. If you will be using a primer other than the universal primers that we provide, please submit at least 5µl per reaction of a properly labeled 3.2pmol/µl stock.
  3. Please note that if more than one PCR product is present in the reaction, your sequence data will be mixed and most likely unusable. We therefore, highly recommend that you check all PCRs on a gel before submission.

Please allow up to a 48 hour turn-around time for data. Samples submitted after 3 PM will not be processed until the following day. If submitted after 3 PM on a Thursday, samples will not be processed until the following Monday.

The facility provides the following Universal Primers free of charge:

  • M13F: 5'-TGT AAA ACG ACG GCC AGT-3'e
  • M13R: 5'-CAG GAA ACA GCT ATG AC-3'
  • T7: 5'-TAA TAC GAC TCA CTA TAG GG-3'
  • T7 Term: 5'-GCT AGT TAT TGG TCA GCG G-3'
  • T3: 5'-ATT AAC CCT CAC TAA AG-3'
  • SP6: 5'-GAT TTA GGT GAC ACT ATA G-3'
  • SK: 5'-CGC TCT AGA ACT AGT GGA TC-3
  • KS: 5'-TCG AGG TCG ACG GTA TC-3'
  • PGEX3': 5'-CCG GGA GCT GCA TGT GTC AGA GG-3'
  • PGEX5': 5'-GGG CTG GCA AGC CAC GTT TGG TG-3'
  • BGH Reverse: 5'-CAA CTA GAA GGC ACA GTC GAG G-3'

Online Submission Instructions

Please allow up to a 48 hour turn-around time for data. Samples submitted after 3 PM will not be processed until the following day. If submitted after 3 PM on a Thursday, samples will not be processed until the following Monday.

Request services by logging onto the OUHSC Core site via our iLab Solutions portal:  https://ouhsc.corefacilities.org/account/login

If you are an internal user, log in using your OUHSC username and password.   If you are an external user and do not already have an iLabs account, please register for an account.  Once you have logged in, please request the service of your choice.  If you have any questions, please email us at microgen_support@ouhsc.edu

 

Analyzing Sequencing Data

 

Finished sequences will be named as follows:

    • date of run+run#_well#_user-initials_sample number on submission form (1,2,3, etc.)_capillary#.filetype
    • example: 2002062101_A01_AB_1_001.ab1.  Even if you submitted your samples in tubes, they will have a well position based off  the way the samples are loaded onto the machine.  Please look for your initials and the sample number following them to line up with the sample number on your form.
    • to identify which sample is which, please look at your form and line up which sample was with which sample number.  This applies if you submitted them in tubes or if you submitted them in a plate.
    • - -
    • for full 96-well plate submissions, the sample names with all be your plate name
  • Please allow up to a 48 hour turn-around time for data. Samples submitted after 3 PM will not be processed until the following day. If submitted after 3 PM on a Thursday, samples will not be processed until the following Monday.

 

Note: Sequences can be viewed by downloading free software at CLC or FinchTV.

 

Pricing

Service OUHSC Non-Academic
Add primer and Capillary Sequence
Primer added by facility personnel to <96 samples submitted in tubes or plates
$6.50/reaction   $16.50/rxn
Capillary sequencing of pre-mixed template and primer
Customer adds primer to template before submission
$6.00/reaction $14.25/reaction
Large Scale Custom DNA Sequencing
For submissions of full 96-well plates
$480.00/plate  $494.00/plate
Qiagen prep of cell pellet 
Does not include the cost of sequencing
$2.75/pellet $6.63/reaction
Qiagen PCR clean up
Does not include the cost of sequencing
$5.50/cleanup  $9.38/cleanup
Small-Scale Fragment Analysis (microsatellite, AFLP, TRFLP and SNP)
Rate applies to submissions of less than 96 samples at a time of fluorescently labeled products for size or allele determination using the ABI 3730xl.  Fragment lengths can range from 35bp to 600bp for microsatellite samples and 15bp to 120bp for SNP samples.
*Please contact us directly if you require facility personnel to perform amplification with fluorescent primers as pricing will be affected.
$5.00/sample $8.88/reaction
High Throughput Fragment Analysis (microsatellite, AFLP, TRFLP and SNP)
Rate applies to submissions of 96 samples at a time of fluorescently labeled PCR products for size or allele determination using the ABI 3730xl.  Fragment lengths can range from 35bp to 600bp for microsatellite samples and 15bp to 120bp for SNP samples.
*Please contact us directly if you require facility personnel to perform amplification with fluorescent primers as pricing will be affected.
$3.00/sample $6.80/reaction