Post in Sanger Sequencing odd data......: ABRF Discussion Forum: ABRF Discussion Forums

Hi Jodie, Since you seem to get better results with the PCR product in a vector, I suspect that the vector is helping to minimize the formation of secondary structure. For DTS (difficult-to-sequence) templates, I generally don't include any additional reagents such as DMSO or betaine. However, I do make several other adjustments which typically break through DTS motifs in the templates: 1) Denaturation temperature is 98C for both the 'hot start' and the cycling steps. For the 'hot start', I generally use only 2 minutes, but you might need longer (perhaps up to 5 minutes) if your product is creating really strong secondary structure. 2) For long templates (>900 nt), I bump the number of cycles from 25 to 35; however, your short product (<400 nt) probably won't benefit from more cycles. 3) For standard cycling, I use only 0.5 ul of BigDye (with 3.5 ul of 2.5X Sequencing Buffer) in a 10 ul reaction; however, for DTS samples, I typically use 1.0 ul of BigDye (with 3.0 ul of 2.5X Sequencing Buffer). The standard ABI reaction would call for 4 ul of BigDye in a 10 ul reaction (8 ul in a 20 ul reaction). For my normal cycling, I've gone to using 95C (10 s), 50C (5 s), 55C (5 s), 60C (just 2 min -- not the standard 4 min). I tested down to 1 minute extension times, and even that usually worked... but not always. When I am working with DTS templates, I do bump the extension time to 2.5 minutes, but these are typically long templates. Given that your product is so short, I would suggest minimizing the extension time as much as possible to minimize the formation of secondary structure... perhaps even dropping to 1.5 minutes. I also tested increasing the extension temperature in the hopes of minimizes secondary structure; however, above 62C, signal strength dropped dramatically... so, 60C seems to be the upper limit for good polymerase activity. Annealing Temperature: I have found very few primers that require staying at 50C for the full 10 s. However, recently, I dealt with some extremely high A/T content samples (and primers), and actually had to drop the annealing temperature to 45C for 45 s (not positive we needed 45 s, but that was the client's PCR annealing temperatures, so we went with it). If your primers have a high annealing temperature, consider bumping the annealing temperature... possibly even to the point of 2-stage cycling (denature at 98C; anneal/extend at 60C)... to help minimize secondary structure formation. To summarize, I would suggest you try the following: 1) Skip the betaine. 2) Increase all denaturation temperatures to 98C. 3) If your BigDye input is very low compared to the ABI protocol, try doubling the amount of BigDye (similar to above). 4) Shorten the annealing time and consider increasing the annealing temperature based upon the Tm of the primer. 5) Shorten the extension time to 2 minutes (possibly even just 1.5 minutes).