Sample Preparation
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Sample Preparation for sequencing:
The most frequent source of problems during a sequencing reaction is bad DNA quality as it directly affects the quality of a sequencing reaction. DNA sequencing is an enzymatic reaction and its polymerase activity can be compromised by the presence of a variety of contaminants including unused primers or dNTP's from the PCR reactions, organics, salts, detergents or incorrect pH in the template solution. This and other common problems will result in poor quality sequence data.
To solicit a quote for Sequencing Services, please fill out the Quote Solicitation Form!
To improve the probability of having the highest quality of data we require a sample submission form from the user. It is completely mandatory.
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We recommend purifying your PCR template using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap) Digestion, Qiaquick column purifications and/or MiniPrep spin columns from Qiagen for plasmid DNA. If using Qiagen columns, please elute with Autoclaved water.
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Please use strip-tubes or 96-well plates to send your samples.
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For PCR products, make a 7.0 uL mixture of:
*15-100ng or 2ng per 100bp
*1.0uL 3uM primer
*bring volume up to 7.0uL with ddH2O
For plasmid DNA, make a 7.0 uL mixture of:
*50-250ng of the insert+vector (miniprep product)
*1.0 uL 3uM primer
*bring up to 7.0uL with ddH2O
For example: If your miniprep product has a concentration of 50ng/uL, your mixture should contain 3.0uL of your miniprep product (to have 150ng in your 7uL mixture), 1.0uL of your primer and 3.0uL of water. You can now ship your mixture to the facility!
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Please contact us for BAC or Lambda DNA (or any other templates larger than 10kb).
*The minimum number of reactions accepted is 15. If sending less; make sure you have read the Terms of Agreement of the SGf.
*Turn-around usually takes 24-72 hours depending on queue. Longer turn-around will always be notified to the user.
*We will gladly purify your product prior sequencing for an additional $20.00 per 96-well plate.
*Positive controls are charged to your invoice.
Sample preparation for fragment analysis:
The most frequent source of problems prior most fragment analysis data collection is the DNA quality and concentration of the PCR reactions as it directly affects your genotyping run since a variety of contaminants such as RNA and proteins may be affecting your reaction. This will result in poor band isolation and low yield bands.
For specific protocols to obtain any source of fragment analysis, please contact facility staff; they will be provided free of charge. We are currently using a ROX based Size Standard for all fragment analysis protocols.
To solicit a quote for any fragment analysis/genotyping services, please fill out the Quote Solicitation Form!
Analysis Software Program availability:
Please visit our Bioinformatics Satellite laboratory (BSL) located at JGD-215. This facility contains all the computing equipment necessary for the analysis of sequencing and genotype data. Be sure to contact SGf personnel for Sequencher Key availability and any other information regarding data analysis.
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SGf is not responsible for data analysis or editing.
Ship samples to:
University of Puerto Rico, Río Piedras
Department of Biology
Julio García Díaz building-Room 214/216
Ave. Ponce de León
PO Box 23360
San Juan, PR 00931
Last modified 2008-04-08 08:32
