Difference between revisions of "Sequencing at Agencourt"

Revision as of 14:10, 22 March 2010

Quicklane Sequencing Service

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Quicklane sequencing provides 24-hour turnaround so that you can receive sample sequences more quickly, for a higher price than slower sequencing services. Submitting miniprepped DNA samples with primers premixed is an available and recommended option. You can always find the complete Sample Preparation and Sample Submission instructions on the Agencourt (now Beckman Coulter Genomics) website.

Sample preparation

• Prepare a 96-well plate or 2D barcoded tubes, provided by Agencourt. Label barcoded tubes with a distinct sample name. Label plates with a distinct name for your sequencing project.
• You must submit 600 - 1000 ng of MP template DNA (mixed with primer) in a final volume of 40 ul for each sequencing reaction. It may be easiest to dry down 600 - 1000 ng DNA and resuspend. Try to submit roughly the same concentration for all samples (15 - 25 ng/ul).
• Add 0.2 pmoles of primer for each sample (if you have 100 uM primer, just dilute it 10X and pipet 2 ul into each tube). If you are sequencing with forward and reverse primers, those are 2 different sequencing reactions, and two different tubes with 1 primer each.
• Resuspend dried DNA and primer in nuclease-free water to a final volume of 40 ul.
• Samples in a well plate must be accompanied by a tab-delimited text file, containing the plate name, well number, and sample name.
• Samples in a well plate can be capped with Optical Cap 8X Strips, or with aluminum adhesive, provided by Agencourt.

Sample Submission

• Click here for sample submission. The sequencing account's username is the professor's full name and the password is the name of the organism studied in Biology111.
• Click "Create a New Project". Give your sequencing project a distinct name.
• Under "Individual Sample Sequencing", select "Quicklane"
• Enter Well ID for each sample, and your given label name under "Sample Name"
• The DNA Template size is the size of the entire plasmid with insert (in basepairs). Enter the Sample Type.
• Check "Primer & Template Premixed" for all samples. You DO NOT have to enter any primer name if you are submitting your own primers premixed.
• Select ".ab1" and ".seq" formats for sequence data.
• Pay with an acquired Purchase Order. The Service Quotation Number, if not already entered, is the one given by the college's sales representative (AGEN #####).
• Include Online Sample Submission and text file with well ID sample names with the samples to ship.
• Package the samples and use Standard Overnight FedEx shipment to mail to: Beckman Coulter Genomics / Attention Genomic Services / 36 Cherry Hill Drive / Danvers, MA 01923
  

Sequencing Glycerol Cell Stocks

SinglePass Plasmid sequencing service allows you to submit a bacterial culture that contains your plasmid to be sequenced, and Agencourt will do the miniprepping, purifying and sequencing for you. You can submit your own primers, or request they use one of their own universal primers for no extra charge. Expect sequence results 4 - 5 days after samples have been received.

This sample preparation will require 12 hrs for growing your cultures, and dry ice for sample shipment. Plan your preparation wisely. You can always find the complete Sample Preparation and Sample Submission instructions on the Agencourt website.

Sample Preparation

• Prepare LB media, 10% glycerol and 50 ug/mL Ampicillin (or other appropriate antibiotic. Other antibiotic concentrations are detailed in the Agencourt Sample Submission guide).
• Aliquot 180 - 190 ul LB-glycerol into wells of a 96-well plate, provided by Agencourt.
• Inoculate cells containing your plasmid to be sequenced into wells. RECORD WELL ID AND SAMPLE NAME.
• Incubate culture plate at 37C for 12 hours, not shaking and loosely covered with cellophane. More than 90% of the filled wells must have grown to be visibly cloudy.
• Prepare primers in separate microfuge tubes. Agencourt uses 20 ul of 3-uM primer in each well. You can send enough undiluted stock primer (100 uM) for the company to dilute for you before aliquoting. Label all primer tubes.
• After 12 hours of cell growth, seal wells with Optical Cap 8X Strips, or with aluminum adhesive, provided by Agencourt. Freeze plate at -80C.

Sample Submission

• Click here for sample submission. The sequencing account's username is the professor's full name and the password is the name of the organism studied in Biology111.
• Click "Create a New Project". Give your sequencing project a distinct name.
• Under "High Throughput Sequencing", select "SinglePass Plasmid Sequencing"
• Enter the number of plates you are submitting, your vector name (ex. pSB1A2), the antibiotic marker, and the number of sequencing directions. Forward and reverse sequencing is Two directions. Check "No" for Are the samples purified?
• Enter the label name and the concentration for each of the primers you are sending in tubes.
• Select ".ab1" and ".seq" formats for sequence data.
• Pay with an acquired Purchase Order; select "Blanket P.O." or submit the Purchase Order amount. The Service Quotation Number, if not already entered, is the one given by the college's sales representative (AGEN #####).
• Fax the Purchase Order to the attention of Genomic Services at 978-867-2601.
• Wrap frozen plate in aluminum foil.
• Place plate in a styrofoam box with dry ice loaded on top and under the plate. Place Online Sample Submission in a plastic bag with the labeled primers.
• Tape box securely. You can tape a FedEx Small Pak envelope to the top and side of the box to hold the shipping label more securely.
• Use Standard Overnight FedEx shipment to mail to: Agencourt Bioscience Corp. / 500 Cummings Center, Suite 2450 / Attention Genomic Services / Beverly, MA 01915