There are several components in a sequencing reaction—template, primer, sequencing reagent, and “other additives.” The mix of these reagents then undergoes 3 steps—cycle sequencing, post sequencing cleanup, and analysis. By examining each of these processes, sometimes in conjunction with controls we provide, we can get you sequencing successfully right away, or, if you’re having difficulty, figure out why you’re not getting the data you desire.
Template is a very common problem area. If your chromatogram is blank, has very low signal, or starts well but gradually dies out, the template should be examined. For plasmids in the 3-10 kb range, 0.2 ug is a good amount of DNA to use. More is almost never better. One problem is that accurate quantification of DNA is not easy. Spectrophotometer readings will invariably overestimate the amount of template DNA, unless you have CsCl banded material. That’s because use of almost all miniprep kits on the market will result in some RNA, chromosomal DNA, and other fluorescent cellular material coming through the purification procedure that will absorb UV light. These other chemicals won’t necessarily inhibit the sequencing reaction, but they will contribute to the A260 reading. As a result, relying on this spec reading alone will cause you to add less DNA than you think (midi and maxi prep kits do a better job, giving a higher plasmid:contaminant ratio). It’s informative to run your template on an agarose gel, using some standard of known concentration and estimate the relative fluorescent intensity following gel staining. We can provide you with a pGEM standard at 0.2 ug/ul to run with your sample. If you are doing many preps and don’t always feel like quantifying your DNA, a reasonable rule of thumb for minipreps of a high copy number plasmid, following lysis of 2-3 mls of a dense overnight culture, is to use 1/10th volume of the eluted sample in the sequencing reaction (but try this first before doing several dozen).
We distribute primers that recognize the Amp or Kan resistance gene present on most commonly used plasmids as a control for template purity. We know these primers sequence well on good templates, so using these in a reaction helps you assess your template “sequencability.” [See http://dna.biotech.wisc.edu/documents/Available_Controls.htm ]
There is a specialized version of the BigDye sequencing reagent that contains dGTP instead of the dITP present in the normal formulation. This is used specifically for templates with problem regions of secondary structure. Such regions are manifested by a chromatogram that looks great up to a certain point, then falls off precipitously. If you are seeing this sort of pattern, we can provide you with an aliquot of the dGTP mix to try. It typically works best in a mixture with the standard BigDye, at a ratio of 3:1 or 2:2 normal:dGTP (it varies from template to template), and should also be used in conjuction with 1M Betaine and elevated extension temperature (68 or 72°). It may take a few attempts to get good data using this reagent. A problems that can occur with the use of dGTP is that “compressions” are evident as peaks that migrate slightly out of register due to annealing within the extension product. Sometimes the sequence needs to be edited manually if the basecaller can’t resolve these patterns.
Other Reaction Components
We provide you with the sequencing buffer shipped by ABI with every enzyme order. Older versions used a 2.5X dilution buffer comprised of 200 mM Tris pH 9.0, 5 mM MgCl2. The new buffer formulation is different and the recipe is a carefully guarded secret. We (and others) have seen that the new buffer does give better results than the old dilution buffer. The new buffer is at 5X strength; the enzyme mix itself is formulated in half buffer, so for a 20 ul reaction you should add 3 ul 5X buffer and 2 ul enzyme mix, 1 ul of which is 5X buffer. This gives 4ul total 5X buffer in 20 ul--perfect! Other commercially available additives are sold as “sequencing reaction enhancers.” Some do work marginally better than the buffer in maintaining signal strength, especially at very low enzyme dilutions (1ul in 20 ul reactions). However, none are cheap, and our calculations indicate no one would save money through using them. However, some users could conceivably benefit. If you’re curious about these, contact us and we can arrange to give you a limited amount of what we have on hand. Also, many companies are willing to distribute a small sample to try. Following that, it would be up to individual labs to purchase what they wanted.
Other non-commercial reagents can work well in getting refractory templates to sequence better. DMSO is a favorite additive of sequencers across the country. We go back and forth here adding itWe use DMSO in 90% of our reactions in house. One issue is we have seen it go bad and actually inhibit the reactions, so you'll want to keep your stocks fresh. But it can help, and it's cheap. Stick with 1 ul/ 20 ul reaction (final concentration of 5%). Also, people have reported the addition of formamide to a final concentration of 5% can help when secondary structure is a problem (a diagnostic chromatogram for secondary structure in a template is good looking data that abruptly terminates)--we have no data on this. As mentioned above in the dGTP secondary structure discussion, a chemical called betaine can enhance sequencing of these abruptly terminating clones, and can also help other template related problems, including nucleotide repeats. Betaine is used by making up a 5M stock, then adding it to a final concentration of 1-2 M (we typically use 1M though some protocols call for more) in your sequencing reaction. It's also commercially available in mol boil grade at 5M concentration from Sigma. Single strand binding protein (SSBP) available from Promega has been reported to help sequencing of refractory secondary structure templates, used at a concentration of 1 ug/20 ul reaction. We have no direct data with this additive.