Core Facilities

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:

*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.