Hints and Tips

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Purity Levels and Applications >>to the top

 

   

The synthesis of oligonucleotides is carried out on a solid support (controlled pore glass) in a series of cycles (number of cycles corresponds to the length of the oligonucleotide) with each cycle comprising a series of chemical reactions (5’-deprotection, activation/coupling, capping and oxidation). A chemical reaction never processes completely. Using phosphoramidite chemistry, the main side reaction is the formation of n-x products due to incomplete activation or coupling, respectively. The longer the sequence, the more n-x products occur. The figure on the right-hand side gives an overview of how the degree of the coupling efficiency (CE) and the length of the oligonucleotide (n) influence the final yield of the raw product (Yield = CEn-1). Thus, the theoretical maximum yield of a full-length product for a 50mer at 99.0% coupling efficiency will be 61% and for a 150mer at  98.5% coupling efficiency only be 11%, respectively. In general, the purity level that you require depends on the effect that alternative sequences (n-1, n-2, … n-x products) will have on your specific experiment. In the following, a short summary is given about the various purification levels that Microsynth offers you as a customer. Furthermore, we specify the required purity level for applications involving unmodified oligos.

 

Desalted Oligonucleotides

All our oligos are at least desalted to largely remove residual low molecular by-products arising and accumulating from the frequent chemical reactions during synthesis. Such purification is sufficient for oligonucleotides shorter than 30 and/or oligonucleotides used for non-critical applications such as PCR, sequencing, probing, mobility shift or hybridization. However, desalted oligos are not recommended for use in molecular cloning projects.

 

HPLC-Purified Oligonucleotides

Oligos <50 bases in length can be well purified via Reverse Phase HPLC. Through this purification approach, preferably residual, n-x truncated oligos (lacking the hydrophobic DMT protection group at the 5’ end) are removed. This results in a 90-95% purity of the targeted oligonucleotide. RP-HPLC is useful for a higher level of purity required for more demanding applications such as cloning, DNA fingerprinting, real-time PCR, FISH, etc.


PAGE-Purified Oligonucleotides

Polyacrylamide gel electrophoresis (PAGE) purification is generally necessary for long oligos (>50 bases) and for all those primers with critical 5' sequences (restriction endonuclease sites, RNA promoters). It is the best method to differentiate full-length oligos from aborted sequences (n-1 oligos), based on size, conformation and charge. PAGE purification has an excellent resolution and yields a product that is, on average, 95-99% pure. In this context, it is important to note that the purity level declines with increasing length of the oligonucleotide, and this is particularly true for oligos >120 bases. PAGE purification is highly recommended for sensitive experiments such as cloning, mutagenesis, DNA fingerprinting, in situ hybridization, gene synthesis, etc.

 

Quality Control

Above all, a stringent quality control system ensures the consistently high quality of all our oligonucleotides. Microsynth performs online trityl monitoring of all oligonucleotides in order to control the coupling efficiency after each cycle. Following synthesis, molecular identity of our oligonucleotides is either checked by MALDI-TOF (up to 50 bases) or by analytical PAGE (51 to 150 bases).

 

 

Handling and Storage of DNA Oligonucleotides
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Handling of DNA Oligos

When handling DNA oligonucleotides, please consider the following:

  • Make sure you use nuclease-free solutions only (see our suggestion below for potential solvents)
  • Handle carefully to avoid bacterial contamination
  • Oligos are generally more stable at higher concentrations
  • Some fluorescent dyes (especially Cy dyes) are especially light sensitive, and exposure to light should be minimized. Furthermore, some dyes may be sensitive to oxidation. Therefore, please ensure that you only open the tube if required.

How to Dissolve Your Oligo?

Generally, oligonucleotides are best dissolved in sterile water or 10 mM Tris-HCl buffer (pH 7.5). However, there are a couple of exceptions that require special attention:

  • Oligos carrying a fluorescent dye should not be dissolved in distilled water. Distilled water usually has a pH of ~6.0 which favors the degradation of the fluorescent dye. In such cases use only 10 mM Tris-HCl buffer at pH 7.5.
  • Oligos carrying fluorescent dyes of the cyanine family such as Cy3 etc. should not be dissolved in 10 mM Tris-HCl buffer at pH 7.5 since they degrade slowly at pH >7.5. In such cases use distilled water only.

How to Re-suspend Your Oligo?

  • Do a short spin at max. speed in a centrifuge to collect the pellet at the bottom of the tube
  • Add an appropriate amount of sterile water or buffer
  • Heat the tube for 5 minutes at 65 °C
  • Vortex or mix by pipetting vigorously up and down

Storage of DNA Oligos

If you want to store oligonucleotides, please consider the following:

  • Please avoid repeated thawing and freezing as physical forces involved may degrade your oligos
  • Depending on your experiment, please choose a suitable storage condition (see our suggestions for potential storage conditions further below)
  • Preparing aliquots may make sense if oligos must be stored and used over a long time period without losing activity

State

Temperature [°C]

Shelf Life

Dried

-25 to -15

Several years

Dried

+15 to +25

5 months up to several years

Liquid

-25 to -15

6 months – 2 years

Liquid

+2 to +8

2 months – 1 years

Liquid

+15 to +25

1 week – 3 months



Handling and Storage of RNA Oligonucleotides
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Handling of RNA Oligos

When handling RNA oligonucleotides, please consider the following:

  • Make sure that you work RNase-free and that you use nuclease-free solutions only (see our suggestion below for potential solvents)
  • Handle carefully to avoid bacterial contamination
  • Oligos are generally more stable at higher concentrations
  • Some fluorescent dyes (especially Cy dyes) are especially sensitive to light and should be stored in light-tight tubes. Furthermore, some dyes may be sensitive to oxidation. Therefore, please ensure that you only open the tube if required.

How to Dissolve Your siRNA or Single-stranded RNA?

Generally, oligonucleotides are best dissolved in sterile RNase-free water or 10 mM Tris-HCl buffer (pH 7.5). However, there are a couple of exceptions that require special attention:

  • Oligos carrying a fluorescent dye should not be dissolved in distilled water. Distilled water usually has a pH of ~6.0 which favors the degradation of the fluorescent dye. In such cases use only 10 mM Tris-HCl buffer at pH 7.5.
  • Oligos carrying fluorescent dyes of the cyanine family such as Cy3 etc. should not be dissolved in 10 mM Tris-HCl buffer at pH 7.5 since they degrade slowly at pH >7.5. In such cases use distilled water only.

How to Re-suspend or Anneal Your siRNA?

siRNA is usually delivered as annealed, purified and ready-to-use duplexes. However, on request we deliver them in dried form as separate strands in two separate tubes. If you don’t know how to anneal, please observe the following protocol:

  • Prepare 100 µM solutions of each RNA strand. Combine 30 µl of each RNA oligo solution and add 15 µl of 5x annealing buffer to reach a final volume of 75 µl. The final concentration of the duplex should be 40 µM.
  • Incubate the solution for 1-2 minutes at 90-95 °C. Then keep this solution at the work bench until it reaches room temperature (cooling should be relatively slow and take about 45-60 minutes). Centrifuge the tube briefly to collect all liquid at the bottom of the tube.
  • Once annealed, duplex siRNA is much more resistant to nucleases than single-stranded RNA and is best stored at -20 °C. The 5x annealing buffer can be freeze-thawed up to 5 times.

How to Re-suspend Single-stranded RNA?

  • Do a short spin at max. speed in a centrifuge to collect the pellet at the bottom of the tube
  • Add an appropriate amount of sterile RNase-free water or buffer
  • Heat the tube for 2-3 minutes at 90 °C
  • Vortex or mix by pipetting vigorously up and down

Precautions against RNase Contamination

RNA is prone to degradation due its free 2'-hydroxy-group. Main causes for degradation are the activity of RNases as well as the prolonged incubation in alkaline solutions. Please observe the following precautions to help keep your RNA intact:

  • Always work with fresh, disposable plastic consumables (e.g. mark a bag of tubes and put it aside. Never grab into the bag, but instead drop the tubes out of the bag. This also applies to pipetman tips). If you must use glassware, be aware that RNases can survive autoclaving – in this case bake your glassware at 250 °C for at least 4 hours.
  • Always wear gloves (RNases are ubiquitous). Change gloves frequently (Be aware that any surface you might touch with your gloves may have already been touched by a person not wearing gloves.).
  • Use RNase-free water/buffers. Most commercially available water is in fact RNase-free. Alternatively, you may produce DEPC-water. DEPC (Diethylpyrocarbonate) reacts with the primary amine-groups and therefore inactivates RNases but cannot be used for e.g. Tris-buffers.

Storage of RNA Oligos

If you want to store oligonucleotides, please consider the following:

  • Please avoid repeated thawing and freezing as physical forces involved may degrade your oligos
  • Depending on your experiment, please choose a suitable storage condition (see our suggestions for potential storage conditions further below)
  • Preparing aliquots may make sense if oligos must be stored and used over a long time period without losing activity

Type of RNA

State

Temperature [°C]

Shelf Life

ssRNA

Liquid

+15 to +25

Varies

ssRNA

Liquid

-25 to -15

Several weeks

ssRNA

Dried

-25 to -15

Several weeks to months

siRNA

Liquid

+15 to +25

Several weeks

siRNA

Liquid

-25 to -15

Several months

siRNA

Dried

-25 to -15

Several months to years

 


Design Guidelines for Primer Probe Sets
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When designing primer probe sets for real-time PCR, please pay attention to the following guidelines.


Design Guidelines for TaqMan Probes
  • Optimal length: 20 bases
  • Length range: 18-30 bases (Length over 30 bases is possible; in this case it is recommended to position the quencher not at the 3’end but internally between the18th and 25th bases counted from the 5’end)
  • Melting temperature (Tm): 68-70°C. Please ensure that the Tm of your probe is 8-10°C higher than the Tm of your primers (8°C for genotyping, 10°C for expression profiling).
  • GC content: 30-80%
  • Avoid runs of an identical nucleotide, especially of 4 or more Gs (otherwise interaction between the nucleotides can cause the oligo to form a secondary structure)
  • Select the strand that gives the probe more Cs than Gs (due to the strong interaction that G displays with itself)
  • Do not put Gs on the 5’end (quenches the fluorophore)
  • When using within multiplex assays (allelic discrimination) consider the following:
    • Position the polymorphism in the center of the probe
    • Adjust the probe length so that both probes have the same Tm
    • If very short probes are needed (less than 18 bases), please inquire

Design Guidelines for Primers

  • Length range: 18-30 bases
  • Melting temperature (Tm): 58-60°C
  • GC content: 30-80%
  • Avoid runs of identical nucleotides, especially of 3 or more Gs or Cs at the 3’end
  • The total number of Gs and Cs in the last five nucleotides at the 3’end of the primer should not exceed two
  • Primers should scan exon-exon junction. Contaminating genomic DNA will not be amplified by these primers
If you encounter any problems, please contact us or just use our Design Service for Primer Probe Sets.


Guidelines for Use of Fluorophores and Quenchers in Real-Time PCR
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Traditionally, FAM-TAMRA is one of the most frequently used pairs for TaqMan probes in which FAM acts as the fluorophore and TAMRA as the quencher. Other quenchers can also be used, especially dark quenchers or black hole quenchers (BHQs) that capture energy from an excited reporter molecule without subsequent emission of light, i.e. they do not fluorescence.

 

Probes made by using dark quenchers tend to be more sensitive in quantitative detection systems, primarily due to lower background fluorescence and a better signal-to-noise ratio than probes that contain fluorescent quenchers. Moreover, dark quenchers enable the use of a wider range of reporter dyes, expanding the options available for multiplexed or genotyping assays.

 

Some fluorophores can be quenched by more than one quencher. In any case the absorption spectrum of the quencher needs to have a good overlap with the emission spectrum of the fluorophore to achieve optimal quenching. Quenchers have a quenching capacity throughout their absorption spectrum, but the performance is best close to the absorption maximum.

 

For multiplex PCR we recommend the following 5’ fluorophore 3’ black hole quencher combinations (are suitable for most qPCR thermo cyclers using TaqMan probes):

  • Channel 1: FAM-BHQ1
  • Channel 2: Yakima Yellow*-BHQ1 (*equivalent to VIC)
  • Channel 3: ATTO550**-BHQ2 (**equivalent to NED)
  • Channel 4: ROX-BHQ2

Recommended final concentrations for standard experiments:

  • Primers: 0.9 pmol/µl (0.9 µM)
  • Probe: 0.2 pmol/µl (0.2 µM)

Recommended real-time-PCR conditions (Tm Primer: 59 °C, Tm Probe: 69 °C):

  • 30 sec 95 °C
  • 30 sec 57 °C
  • 30 sec 72 °C, 35 cycles


 

Design Guidelines for siRNAs
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Our online siRNA design tool helps you design your siRNAs quickly, reliably, and conveniently. It represents a customized tool that combines published design criteria with Microsynth’s own valuable experience. To use our design tool please login first to our webshop and choose siRNA > siRNA Design Tool.

 

Prior to using our siRNA design tool, we recommend you that you acquaint yourself with the following information.

 

General Information

Finding the optimal design strategy of siRNA is an area of active research. Recently, several studies have underlined the importance of several factors for an effective siRNA. Including these (empirically derived) criteria into the design of a siRNA increases the chance of creating a specific and highly effective siRNA in terms of knockdown.

 

Important criteria include the Reynolds Criteria [1, 2]:

  • The GC content of the siRNA should be 30% to 52%.
  • Try to place at least 3 A/Us at position 5-19 of the sense strand.
  • Make sure that there is a lack of internal repeats (Tm of secondary structure < 20°C).
  • Try to place an A at position 19 of the sense strand.
  • Try to place an A base at position 3 of the sense strand.
  • Try to place an U base at position 10 of the sense strand.
  • Try to place a base other than G or C at position 19 of the sense strand.
  • Try to place a base other than G at position 13 of the sense strand.

Note that not all these criteria have to be fulfilled, but generally the more the better. Our online design program sorts the output with a score number that reflects these criteria.

 

Thermodynamic Criteria: Accessibility of the Target mRNA [3]

Secondary structure of the target RNA seems to be important for silencing. Good accessibility (low secondary structure) of the mRNA at the target site increases the chance of successful RNAi.

 

Please Note: 

Many siRNA design software tools indicate mRNA target sequences in the following manners:

  • AA+N19 or
  • NN+N19

The corresponding siRNAs consist then of the N19 core sequence plus an overhang (e.g. N19+dTdT) only. Should you use such a tool and decide to use our synthesis service, please use the N19 core sequence only and eliminate the AA or NN sequences!

 

References and Suggested Literature for the Interested Reader:
  1. Boese et al. (2005). Mechanistic Insights Aid Computational Short Interfering RNA Design. Methods in Enzymology. Vol 392. Pages: 73-96
  2. Reynolds et al. (2004). Rational siRNA design for RNA interference, Nature Biotechnology. Vol 22. Pages: 326-330
  3. Overhoff et al. (2005). Local RNA Target Structure Influences siRNA Efficacy: A Systemic Global Analysis, JMB Vol 348. Pages: 871-881



Questions?
Write an e-mail or call us at +41-71-722 83 33


Related Downloads
Fluorophores and Quenchers.pdf
Guidelines for use of fluorophores and quenchers in real-time PCR

DNA Synthesis and Molecular Biology.pdf
In the present document essential basics about the DNA synthesis and its impacts for molecular biology are given


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