Sanger Sequencing
Decision #1: Standard vs. Difficult vs. Structural 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 exceptional resolution of ~700 bp, with high quality Phred reads of up to 1000 bp.
B. Difficult Template:
The Core will add Difficult Template Buffer (DTB), better known as 5x Combinatorial Enhancer Solution (5x CES), to your sample to facilitate processivity of the AmpliTaq DNA polymerase during cycle sequencing. Common reasons for processivity problems are:
- GC-rich templates, with overall greater than 70% GC content
- GC-rich regions, with greater than 70% GC content (minimum 10-15 bps)
- Homopolymers [such as poly(A) tails]
- Repetitive sequences (such as His tags or polyglutamine)
- Low-quality DNA (such as ancient DNA or DNA obtained via forensics)
- Dirty PCR products
C. Structural Template:
The Core will add twice as much BigDye Terminator (2xBD) to your sample to limit effects of secondary structure on capillary electrophoresis of your DNA during sequencing. Although not often an issue, common secondary structure problems are:
- Hairpin loops
- Stem loops
- Palindromic sequences
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. 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:
- T3
- T7
- T7 Term
- SP6
- M13 Forward (-20)
- M13 Forward (-40)
- M13 Reverse (-24)
- M13 Reverse (-48)
- pGEX 5’
- pGEX 3’
Decision #3: Individual Samples vs. 96 Well Plate
A. Individual Samples
If you are submitting less than 24 samples, you can submit them in individual 1.5 mL microfuge tubes. Please see Sample Requirements for more info. Also, please note that pooling orders whenever possible can result in a price break, as detailed in Pricing.
B. 96 Well Plate
- If you are submitting more than 24 samples, we ask that you please submit them on a 96 well plate. If you submit them as individual samples, we will process them, but we CANNOT guarantee same day service for such orders.
- If you are submitting more than 48 samples, you MUST submit them on a 96 well plate. Please see Sample Requirements for more info. To your benefit, there is a price break for 48+ samples, as detailed in Pricing.
- If you are submitting more than 96 samples, you MUST submit them as more than one order. Please note that splitting the orders such that each is 48+ samples is the most cost effective, as detailed in Pricing.
PCR Cleanup
Please read this disclaimer about PCR product sequencing:
Sequencing a PCR product is much more difficult than sequencing a recombinant plasmid. High-quality sequence requires high-quality template, but PCR reactions are generally contaminated with sequencing inhibitors (like salts and EDTA) or confounding variables (like primers and/or multiple PCR products). Furthermore, determining the actual concentration of template being targeted for sequencing is complicated by this contamination.
A. You Clean the PCR Product
We highly recommend that you clean the PCR product, because you can thoroughly characterize your sample prior to Sanger sequencing
- Confirm there are no primer dimers or confounding PCR products
- Submit the purified template at the appropriate concentration as suggested here
- Choose the appropriate Sanger service type (Standard vs Difficult Template)
- No need to submit sequencing primer separately
B. Core Cleans the PCR Product (i.e. PCR Cleanup / Sequencing)
We aim to be a high-throughput, low-cost service. Characterizing PCR Cleanup samples is time- and cost-prohibitive for the DSF. Nevertheless, our PCR Cleanup SOP does result in quality sequence for many submitted samples. Samples go through the same cleaning process that we use for every cycle sequencing cleanup prior to capillary electrophoresis.
- We do not check for absence of primer dimers or confounding PCR products
- We do not determine template concentration; in fact, we elute all PCR Cleanups in 50 ul of nuclease-free water (i.e. all samples are handled the same)
- We use Standard Template service for all samples
- Keeping track of user-submitted primers reduces efficiency of service
