DNA Sequencing & Genotyping at AWRI/GVSU

Sequencing Submission Guidelines

DOWNLOAD SAMPLE SUBMISSION SHEET

We can perform sequencing reactions on purified PCR products or plasmids.  In addition, you can submit sequencing reactions that you have performed (Ready To Load).

We prefer you to submit samples in 1.6mL tubes.  If you prefer submitting in plate format, please communicate with us beforehand.

Submitting PCR products -($4/sample)

  • PCR products must be purified (see our recommendations)

  • We must know the size and concentration of the PCR product

  • Primers for sequencing must be supplied at 10uM

  • Sequencing submission form must be completed

  • Submit 10uL of your PCR at the following concentrations, depending on size:

 

Size of PCR Product

Concentration to Supply
100-500bp 10ng/uL
500-1000bp 15ng/uL
>1000bp 25ng/uL

 

Submitting plasmids -($5/sample)

  • Plasmids must be purified and suspended in water
     
  • Supply 10uL of purified plasmid at the following concentration (ng/ul) using the formula

Optimally, submit: [40*(Kb of plasmid + insert)](ng/ul)

 Example: 500 bp insert and 2 kb plasmid, send 40*(2 kb + .5 kb) = 100 ng/ul

 Or, submit AT LEAST: [10*(Kb of plasmid + insert)](ng/ul)

  • Contact Ryan or Jeremy if you encounter difficulties purifying your plasmids to the above concentrations
  • Primers for sequencing must be supplied at 10uM
     
  • Sequencing submission form must be completed
     

 

Submitting Ready To Load samples -($2/sample)

  • Ready to load means you have done the sequencing reaction
  • Sequencing reactions must have used ABI's BigDye version 3.1, which is available from Applied Biosystems.  However, you can also purchase small quantities of BigDye from us if you need to.  Contact Ryan Thum or Jeremy Newton for more details.
  • We prefer to purify sequencing reactions; talk to us before doing your own purifications
  • If you require a different sequencing chemistry, talk to us and we will work with you to get your needs met.

 

 

Primers available at the Facility

The Sequencing and Genotyping Facility maintains supplies of these commonly used primers. If you would like to sequence with these primers, you do not need to send an aliquot.

SP6 5’ – TAC GAT TTA GGT GAC ACT ATA G – 3’

T3 5’ – AAT TAA CCC TCA CTA AAG GG – 3’

T7 5’ – TAA TAC GAC TCA CTA TAG GG – 3’

T7 Terminator 5' GCT AGT TAT TGC TCA GCG G  3'

M13 Forward (-20) 5’ – GTA AAA CGA CGG CCA G – 3’

M13 Reverse 5’ – CAG GAA ACA GCT ATG AC – 3’

 

DOWNLOAD SAMPLE SUBMISSION SHEET

Page last modified October 19, 2012