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Our facility has available (at no charge to the client) the universal primers: pUC19 (forward and reverse), T3, T7 and SP6. The sequences to these primers can be found below. Clients can also provide their own primers. A volume of  5 ul of a 5 uM solution per reaction should be submitted. The following recommendations will help in designing optimal sequencing primers:

·       Primer length should be at least 18 bases long to be sure of good hybridization to the template, and longer primers will decrease the risk of binding to more than one site on the template.

 

·       Runs of 4 or more of a single nucleotide, especially guanine, should be avoided.

 

·       Primers with melting temperatures (Tm) above 45° C will produce better results.

 

·       The G-C content should be between 30-80%. If the G-C content is less than 50%, it may be necessary to design a primer longer than 18 bases to keep the Tm above 45°C.

 

·       Make sure the primers do not have a secondary structure, and cannot hybridize to form dimers.

pUC/M13 Forward: 5'-CGC CAG GGT TTT CCC AGT

   CAC GAC-3'

pUC/M13 Reverse: 5'-TCA CAC AGG AAA CAG CTA

   TGA C-3'

 

pUC/M13 Forward(-20):  5’-GTA AAA CGA CGG CCA

    G

 

T3: 5'-ATT AAC CCT CAC TAA AGG GA-3'

T7: 5'-TAA TAC GAC TCA CTA TAG GG-3'

SP6:  5’-ATT TAG GTG ACA CTA TAG