Technical Notes

Tips for Designing Sequencing Primers

Please make sure that your primer can adequately bind to your template. Insufficient primer binding often leads to poor- quality results.
Optimal Sequencing Primer Characteristics:
Approximately 18-24 bases in length
Melting temperature (Tm) between 50 – 60 degrees
GC content should be about 45 – 55%
Have a G or C at 3′ end
3′ end is complementary with your template

Tip: When preparing and using primers, please remember that pmol/µl = µM.

Tips for How to Purify PCR Products

PCR products should be purified by either gel extraction and eluted in water, or by enzymatic treatment. Please use gel extraction if you have more than one product from a PCR reaction (i.e. you have more than one band).
If you have a single PCR band and would like GENEWIZ to perform the PCR clean-up step, simply choose “PCR Product-Un-Purified” from the DNA Types available. Send the same amount of DNA that is required for purified PCR products. Also include a gel image of your PCR product with the volume loaded in each well clearly labeled, and the volume and mass of the ladder.

If you would like to perform the PCR clean-up, we recommend the ExoSAP-IT kit from USB Corporation (Cat. #78200).

Tips on How to Determine Optimum Template Concentration for DNA Sequencing

For plasmids, multiply 10 ng/µl by the length of the template in kilobases.
Optimal plasmid concentration = 10 ng/µl x kb

Example: You have a 4.5 kb plasmid (vector + insert). Multiply 10 times the plasmid size in kb. Thus, the optimal concentration for sequencing is 45 ng/µl. Since we request template in 10 µl, you would submit 450 ng plasmid.

For purified PCR products, multiply 2 ng/µl by the length of the template in kilobases.

Optimal purified PCR product concentration = 2 ng/µl x kb

Example: You have a 700 bp PCR product. Multiply 2 times the product size in kb. Thus, the optimal concentration for sequencing is 1.4 ng/µl. Since we request template in 10 µl, you would submit 14 ng purified PCR product.