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Primer Guidelines
Please use this table as a reference to ensure that your primers will be compatible with our sequencing process.
| Concentration | 1 µM scale (1 pmole/µL), suspended in ddH2O. |
| Volume | 8 µL of primer per reaction |
| Melting Point (Tm) | At least 50°C, 52-56°C is ideal to minimize mispriming. |
| Length | 18-24 bp is ideal |
| G/C content | 50% |
| Dimerization | Primers should not form primer dimers or prime multiple times within your sample. |
| Design | When you design your primers, avoid 3 or more G/C’s in a row, especially at the 3’ end. One G or C at the 3’ end is good. Also, avoid mononucleotide or dinucleotide runs and secondary structures within your primer. Also, primers must prime at only one place in your template, and should not form secondary structures. It is best to choose custom primers with a primer picking program. We offer a primer program here |
| Purification | Desalting is the best value - it is the least expensive and is usually adequate. HPLC is moderately priced, and performs slightly better than desalting. PAGE purification is the most expensive, and should be used if getting the cleanest sequence possible is a priority. |
Stock Primers
To make things easier for our customers, we keep in stock some primers that may be used with at no additional cost. If you are including primer from your own stock and do not want us to use our stock primers with your order, please indicate this in the comments area on the request form. For all orders without comments we will assume that you want us to use primers from our supplied list.
We currently offer:
| Primer Name | Primer Sequence |
| T7 Promoter | TAA TAC GAC TCA CTA TAG GG |
| T7 3’ tag | GGA TCT ACG TAA TAC GAC TCA CTA TAG (for vector pCS2+MT - if other vectors require this promoter region primer, let us know.) |
| T7 Terminator | GCT AGT TAT TGCT CAG CGG |
| T3 | ATT AAC CCT CAC TAA AGG GA |
| SP6 | CGA TTT AGG TGA CAC TAT AG |
| M13 Forward | TGT AAA ACG ACG GCC AGT |
| M13 Reverse | CAG GAA ACA GCT ATG ACC |
| BGHR | TAG AAG GCA CAG TCG AGG CT (note: 3' CT is new in latest batch) |
| SV40 | TTT TTG GAG GCC TAG GCT TTT GCA |
| CMV | CGC AAA TGG GCG GTA GGC GTG |
| GFPf | GGT CCT TCT TGA GTT TGT AAC (note: GFPf=GFPc) |
| EGFPc | CAT GGT CCT GCT GGA GTT CGT G |
| EGFPn | CGT CGC CGT CCA GCT CGA CCA G |
