Click here for complete information on primer design for DNA sequencing. Double-check template the concentrations. PCR fragments are smaller, and thus more effective sequencing templates that are the usual plasmids. Because PCR is intrinsically an exponential process, and because it is usually carried well beyond completion, even rather poor primers will produce amplification in a PCR reaction.
If you have to cycle more than 35 or so times to get an amplification product, or if you have to use unusual additives or odd conditions to achieve success, your primer may not be efficient enough to use for sequencing. Typical laboratory spectrophotometers cannot with any accuracy measure the small amount of DNA that a PCR reaction generates.
Unless you own one of the newer micro-specs e. Please compare them to a reference DNA fragment of similar size and known concentration! You may think that your reaction produced just a single product, but there are very often other things there. Sounds a bit dismal, right? Not necessarily. There are some very good reasons you might want to go ahead and sequence directly from a PCR product. Here are some:. Direct sequencing is much quicker. Common PCR protocols Taq polymerase under standard cycling conditions generate mis-incorporations occasionally about once per 3 kb, in my hands.
If you clone those PCR products and sequence several of them, you will see point mutations in some of the clones. Although many of the individual products have mutated nucleotides, these mutations are scattered randomly and are different for each individual product fragment.
We have many clients who have successfully sequenced thousands — even tens of thousands — of PCR products, with outstanding results. Be critical of the quality of your PCR product and, if necessary, optimize the PCR conditions or gel-purify the desired fragment. Provide 4ul for each reaction. Skip to main content Skip to main content Toggle Search Search. Choose which site to search. Current site. All of UAMS. There are also problems with getting rid of excess primers.
The DNA fragments sequencing reaction products migrate in the capillary during separation depending on their molecular weight more or less proportional to their charge, which is the essence of the method. However, all DNA fragments must be fluorescently labeled terminally, at the 3 'end, each with one fluorophore according to the terminal base , each ACGT base has a different fluorophore and these have different molecular weights.
So, if we had the same DNA fragment identical sequence labeled with different fluorophores, they would have a different molecular weight and migration speed. In practice, we do not have two completely identical fragments with different fluorophores, but two fragments differing in base and having the same or different fluorophores.
This picture shows it nicely:. This is a preview of the raw data before basecalling. About in the middle you see a sequence of 11 red peaks T interrupted by a black one G. The red peaks are evenly spaced because they all have the same fluorescent label and differ in base.
Black also differs in base compared to red to the left or right of it, but has a different fluorophore with a different Mw, in this case lower than the Mw of the red fluorophore , so it migrates in the capillary faster.
In fact, it is a bit ahead, more to the left than we would expect it to be. Therefore, the sequence cannot be read from the raw data or is difficult to. Of course, this effect is removed when analyzing the data:. The migration of fragments peaks in the electrophoretic field is of course always affected by the fluorophore bound to them, and this effect is greater the shorter the DNA fragment the fluorophore thus makes up a higher percentage of the total molecular weight.
The analysis algorithm has to perform peak mobility correction differently if the fragment is say 30 or bases in size, which is a bit of a challenge. The problem of reading short templates can be solved in part by using alternative sequencing chemistry that can read a little better just after the primer kits called BigDye Terminator v1.
However, the typical requirement of a typical client is to sequence as far as possible, about bases, and this sequencing chemistry has a problem with that. You may start reading some ten bases behind the primer instead of twenty, but reading bases will be difficult.
It's just something for something. There is a very nice strategy that solves this situation and combines two approaches, both feasible on the client side. You don't necessarily have to do both, one may be enough, but if you use both, it will have a greater effect.
First, you extend your PCR primers with overhangs compatible to sequencing primers.
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