Difference between revisions of "Davidson Missouri W/CUGI Sequencing"

Revision as of 19:36, 16 July 2009

==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

The sequencing account's password at Clemson is the name of the computer next to the microarray scanner. The user name is Dr. C's email prefix. The website is www.genome.clemson.edu

• You need to send 60 ng of DNA in a tube for each sequencing reaction. Always submit 60 ng for each primer used for sequencing.
• The DNA to be sequenced must be sent dried down, or within 3 µl. Unless you have a good reason not to, always submit the DNA dried down.
• Primers are at 1 pmol/reaction. Unless you have a good reason not to, always submit 3 pmols for each reaction you want performed. Primer solution should be diluted to 1pmol/ul.
• $3.00 / sample
• VR and VF2 melting temperatures are 60 C. 1 pmol/µL = 1 µM.

Note by ATM: I sent 80 ng of DNA for 3 samples and 10 pmol total of primer for 3 reactions.

Sequencing with Agencourt

Quicklane Sequencing

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 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.
• 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.
• 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"
• Select 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 #####).
• 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

Quicklane Sequencing

Sample Preparation:

1.