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| − | ==Sequencing with CUGI: Dr. Campbell's Lab== ftp site is CAMPBELL [at] ftp (dot) genome (dot) clemson (dot) edu {but no spaces in this name} pswrd= 5pFEWyA3 case sensitive | + | ==For Dr. Campbell's Lab== ftp site is CAMPBELL [at] ftp (dot) genome (dot) clemson (dot) edu {but no spaces in this name} pswrd= 5pFEWyA3 case sensitive |
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| | Note by ATM: | | Note by ATM: |
| | I sent 80 ng of DNA for 3 samples and 10 pmol total of primer for 3 reactions. | | I sent 80 ng of DNA for 3 samples and 10 pmol total of primer for 3 reactions. |
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| − | ==Sequencing with Agencourt==
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| − | '''Quicklane Sequencing, Primers Premixed'''
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| − | Sample preparation:
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| − | *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.
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| − | *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 ul DNA.
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| − | *Add 0.2 picomoles of primer for each sample (if you have 100uM primer, just dilute it 10x and pipet 2uL into each tube). If you are sequencing with forward and reverse primers, that is 2 different sequencing reactions, and two different tubes.
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| − | *Resuspend dried DNA and primer in nuclease-free water to a final volume of 40 ul.
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| − | *Samples in a well plate must be accompanied by a tab-delimited text file, containing the plate name, well number, and sample name.
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| − | *Samples in a well plate can be capped with Optical Cap 8X Strips, or with aluminum adhesive, provided by Agencourt.
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| − | Sample Submission:
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| − | *Click [http://www.agencourt.com/sample/ 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.
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| − | *Click "Create a New Project". Give your sequencing project a distinct name.
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| − | *Under "Individual Sample Sequencing", select "Quicklane"
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| − | *Enter Well ID for each sample, and your given label name under "Sample Name"
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| − | *The DNA Template size is the size of the entire plasmid with insert (in basepairs). Enter the Sample Type.
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| − | *Check "Primer & Template Premixed" for all samples. You DO NOT have to enter any primer name if you are submitting your own primers premixed.
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| − | *Select ".ab1" and ".seq" formats for sequence data.
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| − | *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 #####).
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| − | *Include Online Sample Submission and text file with well ID sample names with the samples to ship.
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| − | *Package the samples and use Standard Overnight FedEx shipment to mail to: Agencourt Bioscience Corp. / 500 Cummings Center, Suite 2450 / Attention Genomic Services / Beverly, MA 01915
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| − | '''Sequencing Glycerol Cell Stocks'''
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| − | Sample Preparation:
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| − | *Prepare LB media, 10% glycerol and 50 uM Ampicillin (or other appropriate antibiotic. Other antibiotic concentrations are detailed in the Agencourt Sample Submission guide).
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| − | *Aliquot 180 - 190 ul LB-glycerol into wells of a 96-well plate, provided by Agencourt.
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| − | *Inoculate cells containing your plasmid to be sequenced into wells. RECORD WELL ID AND SAMPLE NAME.
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| − | *Incubate culture plate at 37C for 12 hours, not shaking. More than 90% of the filled wells must have grown to be visibly cloudy.
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| − | *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.
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| − | *After 12 hours of cell growth, seal wells with Optical Cap 8X Strips, or with aluminum adhesive, provided by Agencourt. Freeze plate at -80C.
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| − | Sample Submission:
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| − | *Click [http://www.agencourt.com/sample/ 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.
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| − | *Click "Create a New Project". Give your sequencing project a distinct name.
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| − | *Under "High Throughput Sequencing", select "SinglePass Plasmid Sequencing"
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| − | *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?
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| − | *Enter the label name and the concentration for each of the primers you are sending in tubes.
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| − | *Select ".ab1" and ".seq" formats for sequence data.
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| − | *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 #####).
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| − | *Fax the Purchase Order to the attention of Genomic Services at 978-867-2601.
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| − | *Wrap frozen plate in aluminum foil.
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| − | *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.
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| − | *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.
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| − | *Use Standard Overnight FedEx shipment to mail to: Agencourt Bioscience Corp. / 500 Cummings Center, Suite 2450 / Attention Genomic Services / Beverly, MA 01915
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==For Dr. Campbell's Lab== ftp site is CAMPBELL [at] ftp (dot) genome (dot) clemson (dot) edu {but no spaces in this name} pswrd= 5pFEWyA3 case sensitive
Note by ATM:
I sent 80 ng of DNA for 3 samples and 10 pmol total of primer for 3 reactions.
