Sanger Sequencing

Instruction and Parameters

sanger_sequence specifies which well indices of a container (object) should be sent for sequencing. The wells specified should already contain the DNA to be sequenced and sequencing primer(s) in their respective appropriate concentrations.

{
  "op": "sanger_sequence",
  "type": "standard" | "RCA",
  "object": container,
  "wells": [well_index],
  "primer": container, // only required for RCA
  "dataref": string
}

Two types of sequencing are available, "standard" and "RCA" (rolling circle amplification). Standard sequencing uses Sanger sequencing to sequence DNA templates with appropriate primers. In contrast, specifying "RCA" will result in the use of rolling circle amplification to prepare DNA samples, followed by Sanger sequencing. Samples for RCA sequencing should be submitted as aliquots of bacterial broth with a minimum volume of 30uL.

Device

Strateos currently uses an outside service (Elim) for its sequencing needs.

Each well specified for a "standard" sequencing reaction should have a total volume of 15 µl. We recommend that each of these wells contains the following composition of DNA and primer, premixed together:

Template Type	Amount of DNA	Amount of Primer
Plasmid	500 ng	1 µl of a 20 µM stock
*PCR product	40 ng	1 µl of a 20 µM stock

*It is recommended that PCR products are cleaned up using a method like ExoSap Purification before sequencing.

Each well specified for an "RCA" sequencing reaction should have a total volume of 30 µl, and should contain liquid culture media (with antibiotic) that has been innoculated with a single bacterial clone. Each "RCA" sequencing instruction should also be accompanied by a separate tube containing your primer, as specified in the "primer" field. We recommend that each sample well contains the following composition of bacteria, and that each primer tube contains the following composition of primer (primer and bacteria should be kept separate):

Template Type	Amount of Bacteria	Amount of Primer
Bacterial culture (for RCA sequencing)	30 µl	1 µl of a 20 µM stock

*We recommend that each primer tube contains at least 5 µl of primer, even if it will be used for less than 5 sequencing reactions. We also recommend that each "primer" tube should be prepared as a fresh aliquot, immediately before the sequencing instruction, by making a dilution from your higher concentration primer stocks.

In the returned dataset, sequencing traces will be labelled with the dataref and well id.

More information can be found on the ELIM Biopharm site.