For standard samples supply the Core Facilities with a mixture that contains 12 picomoles of primer, and templates at the concentration listed below, in a final volume of 12 µL in water. If using one of the ten supplied primers, provide listed amount of template in 6 µL water. This is double the amount of template and primer that we actually need so that we can rerun samples if there are any problems.  We may reject samples if supplied otherwise.  More information on primer concentrations below*

DO NOT ASSUME WE WILL DILUTE YOUR SAMPLES TO THE CORRECT CONCENTRATION.
We will run whatever you supply and assume you have diluted correctly.

Concentration of double-stranded template (in water) to supply Core Labs:

PCR Product Vector + Insert
Length

 Total DNA in
Final Volume

 Length Total DNA in
Final Volume
 500 bp 25 ng  3 kb - 5 kb  400 ng
800 bp 50 ng  6 kb - 9 kb  600 ng
1000 bp 125 ng  10 kb - 15 kb 1 μg
2000 bp 250 ng  >15 kb 1.2 μg
3000 bp 350 ng    

*Please consult with Core Facilities staff about amounts of template and primer to use for BAC’s, YAC’s, PAC’s, cosmids, bacterial genomic DNA or M13 preps.

*For degenerate primers, add 12 picomoles primer per degeneracy, but do not exceed 100 picomoles.

Helpful Primer Hints

  • Primers should be at least 18 bases long.
  • Avoid runs of an identical nucleotide, especially guanine.
  • Should be 30-80% GC.
  • Our annealing temperature is 50°C.
  • Avoid primers that have secondary structure or that form dimers.

How To Dilute Your Primers

If you have a primer in solution that is 100 micromolar, that is the same as 100 nmol/ml or 100 pmol/µl. We only need 12 picomoles in one sequencing reaction so dilute in the following method:

  1. Do a 1:10 dilution, take 10 µl of 100 µM solution add 90 µl of water. This will give you a solution of 10 pmol/µl.
  2. Add 1.2 µl of primer to the sequencing mix you supply to Core Facilities to provide us with 12 picomoles.

Core Facilities Primers

We supply the following primers:

Forward Primer (M13F) 5' - TGT AAA ACG ACG GCC AGT - 3'
-95 Forward Primer 5' - GCG GGC CTC TTC GCT ATT AC - 3'
Reverse Primer (M13R) 5' - CAG GAA ACA GCT ATG ACC - 3'
SP6 Primer 5' - ATT TAG GTG ACA CTA TAG - 3'
T7 Promoter Primer 5' - TAA TAC GAC TCA CTA TAG GG - 3'
T7 Terminator Primer 5' - GCT AGT TAT TGC TCA GCG G - 3'
T3 Primer 5' - ATT AAC CCT CAC TAA AGG GA - 3'
Poly-T A-anchored Primer 5' - TTT TTT TTT TTT TTT TTT TA - 3'
Poly-T C-anchored Primer 5' - TTT TTT TTT TTT TTT TTT TC - 3'
Poly-T G-anchored Primer 5' - TTT TTT TTT TTT TTT TTT TG - 3'
 

Turnaround Time

Our turnaround time is generally 2 days for 'standard' orders.  

'Premium' orders are guaranteed a one-business day turn around time when both the order and samples are submitted for processing by 9:00am in the core facility.  Drop samples off at Core Facilities, which is housed in the Agricultural and Life Sciences Building, Room 3012, Oregon State University. Samples go in the "To Do" boxes in the freezer and a web submission form must be submitted.

We are open for drop off from 9:00 am to 5:00 pm.  Sample drop-off is in 3012 Agricultural and Life Sciences (ALS) Building.

Questions should be addressed to Liz Zepeda. Call (541)737-3413 for more information.