Primer Name Primer Express Tm @ 50nM, 50mM NaCl Primer Sequence 5'-3' size (nt) Primer Dimer? (by Primer Express) BGH_Reverse_Long 64.1 ºC ACTAGAAGGCACAGTCGAGGCTGATC 26 Possible BGH_Reverse_Core 61.2 ºC TAGAAGGCACAGTCGAGGCTGAT 23 Not likely BGH_Reverse_Invitrogen 50.6 ºC TAGAAGGCACAGTCGAGG 18 Not likely M13/pUC_Forward 53.7 ºC TGTAAAACGACGGCCAGT 18 Yes M13/pUC_Forward_23 69.8 ºC CGCCAGGGTTTTCCCAGTCACGA 23 Not likely M13/pUC_Reverse 54.1 ºC TCACACAGGAAACAGCTATGAC 22 Not likely M13/pUC_Reverse_GC+ 54.3 ºC GCTATGACCATGATTACGCC 20 Not likely SP6_Promega 35.5 ºC TATTTAGGTGACACTATAG 19 Not likely SP6_NEB_24 47.0 ºC CATACGATTTAGGTGACACTATAG 24 Not likely SP6_Core 43.5 ºC ATTTAGGTGACACTATAGAATAC 23 Not likely T7 Promoter (Promega) 43.4 ºC TAATACGACTCACTATAGGG 20 Possible T7 Promoter Neo (Core) 38.4 ºC TAATACGACTCACTATAGG 19 Not likely T7_Promoter_EEV_Promega AAGGCTAGAGTACTTAATACGA 22 T7_Terminator 52.6 ºC GCTAGTTATTGCTCAGCGG 19 Possible T3_Promega 49.4 ºC ATTAACCCTCACTAAAGGGA 20 Not likely
- BGH primers anneal to pKPPURO, pNKTV1907, pcDNA3, pcDNA3.1, pcDNA3.1/Neo, pCR3.1, pIRE51hyg, pIRE51 neo
- M13R/pUC Reverse (=M13R) primer does have some homology with common vectors at the 3' end and can produce some background.
- M13/pUC_Reverse_GC+ (M13R GC+) primer was designed to avoid this problem and works with most pUC-, pGEM-, and pBluescript-derived plasmids. However, there are some vectors on which it will not work due to different sequence at 3’ and / or 5’ ends
- M13 R primer anneals to some plasmids at the primers 3’ end 16 bp or greater, but not the full length.
- SP6 primer does not match with sequence at the 3’ end of the SP6 genome perfectly. Therefore, the different SP6 primers do not match all vectors with "SP6" primer sites as the sequences differ slightly.
- T7 promoter Promega primer does not match 8 out of many other plasmids at the 3’ end.
- T7 promoter Neo (core) primer can be used to sequence both the vectors with normal T7 promoter sequence and pCIneo-derived vectors with the shorter T7 sequence which have two Gs at 3' end; whereas T7 primers has three Gs.
- T7 promoter EEV Promega is utilized by Promega to sequence pCIneo derived vectors and the inserts they carry.
- Invitrogen pCRII vector (had SP6 and T7 primer sites). Due to a Promega patent on the SP6 sequence, Invitrogen had to remove the SP6 site to make the pCR2.1 vector, which does not have an SP6 site.
Before specifying a particular primer for DNA sequencing, verify the primer site sequence is present in your vector.