Agencourt Sequencing

–Draft– Don’t trust these yet

Full instructions from Agencourt can be found here,with individual PDFs for “Getting Started“, “Sample Prep“, “Sample Submission” and “Shipping

Amplified Template DNA

  1. Send at least 20ul of PCR product (Note: we do not have to clean PCR products anymore)
  2. Use a full-skirted plate (use ABgene plates in the labeled box in the bottom drawer of Julienne’s lab bench)
  3. Standardize concentration across the plate (I think the minimum is 18ng/ul) using rough quantification using the ladder
  4. Label plate with the unique name and seal with film.

Primers

  1. IF you are using just one forward and one reverse primer for your plate you can send 100µL of 100µM of each in an Eppendorf tube
  2. ELSE you need need to send two additional plates one each with the forward and reverse primers
    1. Array primers into to the wells that correspond to the template.
    2. Add 20µL of 5µM primer to each well.
    3. Label each plate with same name as template plate plus FPrimers or RPrimers.
    4. Seal each primer plate with film.

Datasheet

  1. Download a blank datasheet [here] for each plate of sequencing
  2. Rename the file with the name you used on your template and primer plates
  3. Enter sample names and primers and primer sequence
  4. Enter additional Primer info in the right hand panel.  A filled out example is [here].
  5. Submit your order online [here]

Username: ***on whiteboard***

Password:  ***on whiteboard***

Shipping

  1. FedEx pickup is at 3 PM, make sure everything is organized ahead of that.
  2. By noon on the day you are going to send samples out you need to alert Cal before hand that you want to send a package overnight delivery, and tell her the address (below) so she can organize it for you.  Do this by sending him an email with the following information:
  • The number of packages to be sent (usually 1 – multiple plates can be sent in one box)
  • The funding source to pay for shipping, the code for Rich’s NSF grant is RADIATE
  • The address to ship to:
Beckman Coulter Genomics
Attention Genomic Services
36 Cherry Hill Drive
Danvers, MA  01923-257

3. Get a box from Cal, and pack it with dry ice completely around your plate (get key from Cal who’ll also direct you to where to get it).

Beckman will email you when the data are ready, they post data for a limited time so be sure to get your sequences promptly.  You can collect your data from the FTP: https://cust.agencourt.com

The username and password are the same as the order submission site.

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