Decision #1: Standard vs. Difficult Template
A. Standard Template
Most plasmid samples would be considered standard. Our Sanger sequencing SOP (standard operating procedure) is designed to work with miniprep-quality recombinant plasmid DNA and a high-specificity sequencing primer to give contiguous reads >800 bases and over 1000 high-quality (Q>20) Phred calls
B. Difficult Template
We will add our proprietary Difficult Template Buffer (DTB) to your sample to facilitate template denaturation and processivity of the AmpliTaq DNA polymerase during cycle sequencing. Common reasons for processivity problems are:
- GC-rich templates, with overall greater than 60-65% GC content
- GC-rich regions, with greater than 60-65% GC content (minimum 100-150 bp)
- Homopolymers [such as poly(A) tails]
- Repetitive sequences (di- or tri-nucleotide repeats, such as His tags)
Decision #2: My Primer vs. Core Primer
A. My Primer
You supply a primer for cycle sequencing. YOU MUST ADD THE PRIMER TO YOUR SAMPLE (aka pre-mixed).
We reserve the right to delay or refuse orders with the primer supplied separately.
B. Core Primer
We can supply any of these 10 common MCS primers for cycle sequencing:
- T7 Promoter
- T7 Terminator
- pGEX 5’
- SP6 Promoter
- M13 Forward (-20)
- M13 Forward (-41)
- M13 Reverse (-27)
M13 Reverse (-48)
- 5'-AAA TTA ACC CTC ACT AAA GG-3'
- 5'-TAA TAC GAC TCA CTA TAG GG-3'
- 5'-GCT AGT TAT TGC TCA GCG GT-3'
- 5'-GGG CTG GCA AGC CAC GTT TGG TG-3'
- 5'-CCG GGA GCT GCA TGT GTC AGA GG-3'
- 5'-GAT TTA GGT GAC ACT ATA G-3'
- 5'-GTA AAA CGA CGG CCA GT-3'
- 5'-GTT TTC CCA GTC ACG AC-3'
- 5'-GGA AAC AGC TAT GAC CAT G-3'
- 5'-AGC GGA TAA CAA TTT CAC ACA GGA-3'
Decision #3: Tubes vs. 96-Well Plate
B. 96-Well Plate
- Preferred for 24-47 samples
- Required for 48+ samples (includes price break)
- Please use PCR plates with conical wells
- See Sample Requirements for more details
For 24-47 samples, we CAN'T guarantee same day service for individual samples because it slows down our workflow.
PCR Cleanup + Sanger Sequencing
PLEASE NOTE: We offer a "one-size-fits-all" protocol for PCR cleanup with no quantification. Although the below relationship between amplicon size and cycle number is generally successful with our SOP, inherent variability among PCR samples often requires empirical optimization (via reruns) at cost to the customer. Thus, we highly recommend that you clean your PCR product and thoroughly analyze it via gel electrophoresis and fluorometry prior to Sanger sequencing. Recommended concentrations can be found in Template Prep.
- 200-500 bp amplicons, 12-15 cycles of PCR
- 500-1000 bp amplicons, 15-18 cycles of PCR
- >1000 bp amplicons, 18-22 cycles of PCR
PLEASE NOTE: These recommendations are based on PCR amplification from ~100 pg of pure plasmid DNA.
Although rare, sometimes DTB and/or our standard Sanger protocol are not sufficient for sequencing a DNA template. We offer custom sequencing for these very difficult templates, which generally involves alternative sequencing chemistry or other modifications to our SOP. We are happy to consult with you regarding custom sequencing at no charge. However, we do charge a higher fee for custom sequencing to cover the cost of the specialized reagents (currently $5.75/sample for UT). Examples:
- Problematic secondary structure (such as hairpin loops, stem loops, or palindromic sequences)
- Sequencing directly from gDNA
- PCR amplicons <200 bp or vectors larger than 15 Kb