Genomics Core Facility

Sanger Sequencing Guidelines

Below are guidelines for submitting sequencing samples for Individual Sanger Sequencing or 96 well plate Sanger Sequencing:

Individual Sanger Sequencing

Place Individual Sanger Sequencing orders on the dnaLIMS site by clicking on the Enter Individual DNA sequencing requests link.

DNA Templates

Submit 5 μls of template per reaction, and at the following concentrations:

  • 20 ng/μl for PCR products up to 400 bases
  • 40 ng/μl for PCR products 400-1000 bases
  • 200 to 400 ng/μl for plasmids up to 10K
  • 400 ng/μl or greater for plasmids 10K-100K

Primers

Submit 5 μls of primer per reaction at a concentration of 1 μM

These Sequencing primers are supplied by the facility free of charge:

Standard Primer Information

Primer

Sequence

Tm

T7

TAA TAC GAC TCA CTA TAG GG

60

Poly-dT

A

60

T3

ATT AAC CCT CAC TAA AGG GA

na

T7-term

GCT AGT TAT TGC TCA GCG G

60

SP6

CGA TTT AGG TGA CAC TAT AG

60

Gal4AD

TAC CAC TAC AAT GGA TG

60

Gal4BD

TCA TCG GAA GAG AGT AG

60

M13

TGT AAA ACG ACG GCC AGT

60

M13-40

GTT TTC CCA GTC ACG AC

60

M13-47

CGC CAG GGT TTT CCC AGT C

60

M13R

CAG GAA ACA GCT ATG ACC

60

M13R-48

AGC GGA TAA CAA TTT CAC A

60

BGHrevprim

TAGAAGGCACAGTCGAGG

na

Drop-off samples in accordance with the sample drop-off guidelines.

Samples and primers must be submitted in at least 0.6 ml tubes, strip tubes will not be accepted.

Submitting 96 well Plates for Sequencing

  1. To submit plates for sequencing you will need to purchase non-skirted 96 well PCR plates and plate seals. 
  2. Add 2 uls of template at the following concentrations to the plate (note: this is only for plates, not for regular submissions):
    • Plasmid DNA: 200-400 ng/μl
    • PCR up to 400 bases: 20 ng/μl
    • PCR 400-1000 bases: 40 ng/μl
  3. Add 2 uls of primer at a concentration of 1 uM.  If using a primer supplied by the facility, make note in the comment section of the order that the primer needs to be added. 
  4. Submit samples on the LIMS System using the Upload and Import File Feature. 
    • Follow the instructions to download the file.
    • Type in sample and primer names.
    • Upload the file and print out a copy of the order to submit with the plate.

Things to remember when submitting plates:

  • If multiple primers need to be added to the plate, place samples with the same primer in one section of the plate. For example, place all samples that need T7 in one area of the plate and all samples that need SP6 in another area of the plate.
  • Label the plate with the PI name and the Order Number.  Label the plate, NOT just the seal.
  • Make sure an order form is printed out and submitted with each plate.
  • When loading the plate, start at A1 and go down to B1, C1, D1, etc. and then go up to A2. Do not go across the plate because this will cause the sample names to be imported incorrectly. If there are skipped wells (wells with no sample in them), make sure to put something in for the sample name (such as blank or empty). If not, this will cause all the sample names to be imported incorrectly.
  • Drop off the plate in accordance with the sample drop-off guidelines on the black desk outside Room 413 Chandlee between 8:00 a.m. and 4:30 p.m.
  • Plates are not guaranteed to be run the same day that they are submitted.