UCR

Institute for Integrative Genome Biology



DNA Fragment Analysis Services


Genomics Services

DNA FRAGMENT ANALYSIS SERVICES

The Genomics Core provides DNA fragment analysis services including microsatellites, single nucleotide polymorphism (SNP), and single stranded conformation polymorphism (SSCP) using the ABI 3130XL Genetic Analyzer. Protocols provided are for microsatellite marker analysis.  Please contact the Genomics Core staff by email (gencore@ucr.edu) or phone (951) 827-3984 if you plan to use these services.

Protocols

Preparation of DNA Templates

Genomic DNA can be purified using either your own methods or commercial kits such as DNeasy kits from QIAGEN.  It is important to measure DNA concentrations and quality which can be done easily using the Nanodrop ND-1000 or 2000c Spectrophotometers or the Qubit Fluorescence Reader. We recommend that your DNA concentration be in the range of 50—100 ng/ml.

Primer Synthesis

Our spectral matrix is based on dye set DS-33. This dye set allows us to label primers with four different fluorescence dyes: 6-FAM, VIC, NED and PET. Only one primer (either forward or reverse) needs to be labeled with fluorescent dye at the 5’ end.  You can order custom primers from Applied Biosystems or other vendors such as SigmaGenosys. But please keep in mind that VIC, NED or PET labeled primers may be available only from Applied Biosystems due to intellectual property issues. You can also label primer with HEX instead of VIC.  The following table indicates color colors as shown in an electropheragram.


Dye

Color

6-FAM

Blue

VIC/HEX

Green

NED

Yellow

PET

Red

LIZ (size standard)

Orange

PCR Setup

Typically PCR conditions must be optimized in order to obtain successful amplification. The following reaction protocol and thermocycler conditions can be used as a start if you do not have one already.


Component

  Volume (ml)

Water

6.0

MgCl2 (25mM)

0.5

dNTPs (2mM)

1.2

10 x PCR buffer (with 15 mM MgCl2)

1.2

Forward primer (labeled) (10mM = 10pmol/ml)

0.5

Reverse primer (10mM = 10pmol/ml)

0.5

Taq polymerase (5 units/ml)

0.1

Template DNA (10ng/ml )

2.0

Thermocycler conditions: 95 oC  3’ (1x); 94 oC  45’’,  55oC 45”,  72oC 1’ (total of 34 cycles); 72oC 10’, hold at 4oC.

Post-PCR Multiplex

PCR products can be multiplexed in order to reduce cost. PCR products amplified with different dye-labeled primers can be combined at equal or different ratios depending on the fluorescence signal strength. We strongly recommend that you conduct a pilot experiment before doing large-scale multiplexing. This may ultimately reduce your costs due to failed experiments. Please contact the Genomic Core staff if you have any questions.

Note: Our policy is to charge for each sample run which is based upon reagent costs and labor. This includes samples run as part of optimization experiments.

Submission of PCR Products for Fragment Analysis by ABI3130XL

When submitting samples, fill in the online submission form completely. Submit your PCR products (with or without multiplexing) in a 96-well plate that is covered with aluminum foil to the Genomic Core. This is available at the IIGB Billing and Reservation site. If you are not registered, this will be required to access the form. Submit your PCR products (with or without multiplexing) in a 96-well plate that is covered with aluminum foil to minimize light exposure to the Genomics Core (2016 Keen Hall). You may also submit your sample ID files as an Excel file to the Genomic Core Staff (gencore@ucr.edu). We will mix your samples with formamide and size standards, and then load them onto ABI3130XL. Our current capacity is 384 samples per 24 hours. In most cases, you will receive raw data the business day after we receive your PCR products. Please keep in mind that we strive to meet this turn-around time; however, depending upon our workload your results may be delayed. Your raw data will be provided via an email containing a Dropbox link to your Dropbox account. You are not required to have a Dropbox account to open the link and download your data.

Data Analysis with GeneMapper 4.1

Raw data extracted from the ABI3130XL must be analyzed with GeneMapper software. Please visit the Microsatellite Analysis Getting Started Guide  for GeneMapper v4 for more information. The Genomics Core provides individual training in the use of GeneMapper 4.1 and can be requested by contacting gencore@ucr.edu.


More Information

General Campus Information

University of California, Riverside
900 University Ave.
Riverside, CA 92521
Tel: (951) 827-1012

Career OpportunitiesUCR Libraries
Campus StatusDirections to UCR

Genomics Information

Institute of Integrative Genome Biology
2150 Batchelor Hall

Tel: (951) 827-7177
E-mail: Aurelia Espinoza, Managing Director

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