Required DNA Concentrations for Automated Sequencing
**The following reflects the required template and primer concentrations for ALL samples arriving for processing at the UNT DNA Core Facility:
WARNING!!! Primer concentrations are given in 'pMol/µl' --- That is Picomoles per Microliter, NOT PICOMOLAR!
| Template Type | Template Conc. | Template Amt. | Primer Conc. | Primer Amt. |
|---|---|---|---|---|
PCR Product (100-200 bp) |
1-3 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
PCR Product (200-500 bp) |
3-10 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
PCR Product (500-1000 bp) |
5-20 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
PCR Product (1000-2000 bp) |
10-40 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
PCR Product (2000+ bp) |
20-50 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
Genomic DNA (single-stranded) |
20-50 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
Genomic DNA (double-stranded) |
150-300 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
Plasmid (mini-preps, maxi-preps, 4-15 kb) |
50 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
Plasmid (PCR product) |
1-3 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
10 µl per rxn |
siRNA construct |
200 ng/µl |
10 µl per rxn |
3.2 pMol/µl |
20 µl per rxn |
Large DNA (plasmids over ~20 kb, cosmids)* |
500 ng/µl |
10 µl per rxn |
30 pMol/µl (30uM) |
20 µl per rxn |
For EACH sequencing reaction we need 10 µl of template and 10 or 20 µl of primer, in separate tubes. If one sample (template or primer) is to be used in multiple reactions, multiply the volume accordingly. All components should be dissolved in distilled water only!!!
* Experimental. Talk to Core Director (jajohnson@unt.edu)
At the concentrations listed above, we will use between 3 to 4 µl per reaction. We request 10µl per reaction in case we need to do a second run without having to contact you for additional template or primer.
Back to Sample Preparation and Submission Page