Sanger sequencing

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Low template concentration – What can you do?

For successful sequencing analysis it is crucial to use optimal template concentration (as described in our guidelines for sample preparation). If you do not have required amount of template, the total volume of a sample can be lowered but the template to primer ratio must be kept. The problem of low template concentration cannot definitely be solved by only increasing the total sample volume.

In this context, it is also important to mention that a spectrophotometer (Nanodrop) is not a suitable tool for accurate quantification of DNA template (especially at lower concentration range). Primers, proteins, as well as RNA absorb at 260 nm and their presence therefore leads to overestimation of DNA concentration. For this reason we recommend agarose gels, Qubit or some other fluorescent based methodology to measure template concentration.

When preparing a sample, the first important step is to choose an appropriate method for isolation/purification of DNA and/or optimize the PCR conditions to achieve sufficient amounts of DNA templates. If however you already have your sample prepared and its final yield is insufficient, there are some ways how to try to concentrate it up.

  • SpeedVac - If you have such a device in your lab, it is very simple and user-friendly method with good results (no loss of DNA compared to other methods). On the other hand, you may end up also with increase of the concentration of salts (especially when the sample was eluted using some buffer) as well as some other potential inhibitors that may negatively affect downstream applications including DNA sequencing.
  • Evaporation - Similarly, reducing the volume of a sample can be achieved by incubating the sample in a thermoblock/ PCR cycler (in tubes with open lids, at e.g. 58°C). Time of incubation must be selected based on the sample initial volume. Here, again, remember that this will also concentrate all the components of a sample (including salts and other contaminants).
  • Magnetic Bead-Based Method (e.g. Agencourt AMPure XP) - We can recommend this method for the high efficiency of DNA recovery. The final volume can be reduced even several times (e.g. 10x). The advantage of this method is that only the DNA is concentrated and therefore it is also useful if you want to purify your sample from potential inhibitors. Be careful to optimally dry out the last remnants of ethanol - when the beads are overdried, the elution efficiency can significantly decrease leading to some loss of DNA (especially of larger fragments).
  • DNA precipitation - Most commonly, DNA is precipitated using ice-cold ethanol, eventually isopropanol if the total volume of a sample should be kept at minimum (isopropanol is however less volatile and when it is not removed completely, it may inhibit sequencing reaction). In principle, the DNA sample is resuspended in lower volume than you started with and any possible non-DNA contamination in your sample can be also removed. Since some amount of template will definitely be lost when using precipitation, you better think twice when your DNA amounts are limited. The longer precipitation and centrifugation time may help.
  • Column based kits - Typically, lower elution buffer volumes added to the DNA binding membrane produce lower yields but result in higher concentrations. Modifications leading to higher DNA yield can include: heat the elution buffer up to 70°C; after applying the elution buffer to the column spin it first at very low speed (around 50 g) (which should ensure that the elution buffer reaches every part of the membrane); extend incubation time (5-10 minutes) before final high-speed centrifugation to collect the sample. However, it is also important to realize that the lower the volume of elution buffer, the higher the concentration of contaminants after elution.

In summary, it should be noted that these methods are to be used to solve problems with valuable samples and are not suitable for routine use.

 

Martina Kujanová, info@seqme.eu

 

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