DNA/RNA Synthesis

FAQs - Oligo Synthesis and Specialty Oligos

  1. Why are there no oligos seen in the tubes?
  2. How do I reconstitute my lyophilized oligo?Why do I observe liquids in my oligo tube?
  3. Do I use TE buffer or DI water?
  4. How do I store my oligos?
  5. How do I store and handle my fluorogenic Oligos?
  6. Why is there liquid in my tube of oligo?
  7. My one-year old, properly stored oligos seem to have degraded!
  8. How does 1st BASE calculate the Tm of an oligo? Why does this value differs from my own primer software?
  9. Why does the final yield I obtain differs from the synthesis scale that I had indicated? How do I know which synthesis scale to order?
  10. Is PCR grade pure enought for routine PCR and sequencing?
  11. What is TOP purification and what can it be used for?
  12. Why is there no amplification with my primers?
  13. What is carry-over contamination and how can I prevent it?
  14. What Quality Control steps does 1st BASE perform?
  15. Why are there "mutations" in my oligo?
  16. What are these mutations exactly?
  17. Will I eliminate mutations with purified Oligos?
  18. Can you make the oligos having a high percentage of "G" residues?
  19. Why can't i clone double stranded Oligo directly?
  20. How many bases are needed before restriction sites on the 5'-ends of oligos?
  21. What extras does 1st BASE give?
  22. I need help with the design of siRNA duplexes or Dual-labeled Fluorogenic Probes, would 1st BASE be able to help me?
  23. What guarantees come with 1st BASE's proposed probe / siRNA designs?

 

FAQs - Gene Synthesis

  1. What is the difference between custom oligos and a synthetic gene?
  2. What Quality Control steps does 1st BASE include to ensure 100% fidelity?

 

Answers - Oligo Synthesis and Specialty Oligos

  1. Why are there no oligos seen in the tubes?

    Oligos are delivered either Iyophilized or in buffer/water in a concentration of 100 µM.

    in Iyophilized form, DNA is translucent and thus not wisible to the naked eye. Depending on how the Oligos is dried, it could appear as a clear film at the bottom of the tube(not apparent to the naked eye).

    Sometime, it could appear as puffy pellet. The puffy pellet is a result of formation of ice during the final dry-drown process where all the water is eventually dried off, leaving the oligos to retain the structure of ice crystals.

    Regardless of how the oligos appear in the tube, you need to re-suspend them before using. Always spin-down the tubes before opening as the oligos could be dislodged during transportation.

  2. How do I reconstitute my lyophilized oligo?Why do I observe liquids in my oligo tube?

    Our oligos are delivered lyophilized for improved stability. During lyophilization, DNA may become powdery and can easily fall out of the tube, so always spin the tube's contents down before opening each tube. Prior to using lyophilized oligos, reconstitute them in TE buffer or DI water to an appropriate concentration. For convenience, we suggest a concentration of 100 µM. Please refer to our datasheet on the volume of buffer or water to add to the lyophilized oligos to achieve this concentration. Dissolve by vortexing. Do not mix by pipetting up and down. This can lead to contamination of your pipette shaft.

  3. Do I use TE buffer or DI water?

    Either can be used though DI water is increasing favored now for the simple fact that EDTA from TE buffer inhibits PCR, where oligos are most widely employed. DI water on the other hand would require careful handling which is asily achieved through proper sterile technique.

    Under acidic conditions, DNA oligos can become depurinated. On the other hand, the phosphodiester bond of RNA oligos can be hydrolized under basic conditions.

  4. How do I store my oligos?

    Oligos are chemically stable. If stored dry (lyophilized), they will be stable for years. However upon hydration, they are susceptible to degradation by nucleases. Even so, hydrated oligos, if handled correctly, should still be stable for years. Oligos can undergo degradation from exposure to low pH (<3) or heat, leading to depurination and cleavage. Any oligo can be degraded by contaminating environmental nucleases such as microbial or fingertip nucleases.

    You can store hydrated oligos by refrigeration or freezing but refrigeration is increasingly preferred. Refrigeration avoids freeze-thaw cycles and offers convenience. Even when oligos are stored in DI water and refrigerated, they have been observed to be stable for over two years.

    When freezing is preferred, it is recommended to aliquot stock concentrates to several tubes.

  5. How do I store and handle my fluorogenic Oligos?

    If exposed to light fluorogenic oligos are more fragile than unmodified oligos and their fluorescence intensity will decrease over time. To maintain their fluorescence efficiency, fluorogenic oligos should be stored in the dark at -20°C. Since Cy3 and Cy5 can be decomposed at a pH above 9, they should not be stored under basic conditions.

  6. Why is there liquid in my tube of oligo?

    If you had indicated for the oligos to be delivered in 100µM concentration, we will reconstitute the oligos using the volume indicated on the oligo datasheet. This is why you would observe liquids in your tubes. This service is not extended to international orders.

  7. My one-year old, properly stored oligos seem to have degraded!

    This was widely reported late in the 1990s among several research laboratories worldwide. Please be assured, this is not the result of poor quality oligos or improper storage. The often held conclusion is contamination of primer stocks from heavily built up contamination in pipette shafts. Strictly PCR laboratories do not witness this phenomenon frequently. What they did differently from research laboratories is the separation of PCR only laboratory areas, the use of aerosol filter tips and positive displacement pipettes. For research laboratories, such precautions can be a luxury. The least research laboratories can do is use aerosol filter tips for PCR reaction setup coupled with PCR only pipettes to control contamination. Finally, it helps a lot not to store post-PCR products in the same fridge/freezer with the primers or PCR reagents.

  8. How does 1st BASE calculate the Tm of an oligo? Why does the Tm value from my own primer software differ from the Tm reading on the datasheet?

    We use two standard approximation calculations. For sequences less than 14 nucleotides the formula is:
    Tm= (wA+xT) * 2 + (yG+zC) * 4
    where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.

    For sequences longer than 14 nucleotides, the equation used is (Wallace Rule):
    Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

    ASSUMPTIONS:
    Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0.

    There are up to 9 different algorithms used to calculate the Tm of oligos and the different Tm values does not indicate that the primers will different from one oligo service provider to another. Simply use the same algorithm or software to optimize the Tm of your oligos. Then perform your experiments as you designed it to be. In the process of synthesis, we don't change anything with regards to the Tm of the oligos and the difference shown in the datasheet is just a matter of different ways of calculations.

  9. Why does the final yield I obtain differs from the synthesis scale that I had indicated? How do I know which synthesis scale to order?

    The synthesis scale is based on the amount of the first base attached to the solid support to start the oligo synthesis. For larger scales, the amount of solid support is increased. It is NOT the expected final yield. The yield depends on the size of the oligo, the coupling efficiency, and the base composition. Oligo synthesis involves many steps, including:

    • synthesis
    • cleavage from solid support
    • deprotection, and
    • desalting purification

    Decreasing yield as the length of the oligo increases

    All the synthesis steps involve an increasing inefficiency as the oligo length is increased. Additional purification such as PAGE or HPLC will also result in the loss of some product. Therefore, synthesis inefficiency coupled with losses from purification, the quantity of oligo ultimately received is always lower than the theoretical yield.

    To decide which scale synthesis to order, first determine the amount of oligo required (no. of PCR reactions to carry out). After that, compare required yield with the yield guaranteed for the scale. Please note that modifications (if any), and purification will reduce the final yield due to increased processing. As such, order such oligos at a higher synthesis scale.

    For most PCR and sequencing needs, only a minute amount of oligo is needed. For example, the majority of sequencing protocols call for 10 picomoles of primer. For an average 25-mer oligonucleotide, 1 OD260 unit is equal to about 4 nanomoles, or enough primer to do 400 PCR or sequencing reactions. Unless a very large number of reactions are planned using a given primer, it is seldom necessary to order more than a 50 nmole scale primer.

    At 1st BASE, our guaranteed amount of oligos are as follows:

    Scale Guaranteed Amount of Oligo
    50 nmole scale, PCR Grade 2 - 3 OD
    200 nmole scale, PCR Grade 10 OD
    1umole scale, PCR Grade 40 OD
    PAGE Purified Oligos 1 OD
    Modified Oligos + TOP purified 3 OD

    Note: Above mentioned final yield are applicable to Oligos up to 50mers only. For longer Oligos, please enquire.


  10. Is PCR grade pure enought for routine PCR and sequencing?

    Purification options incur additional cost to oligos. RCP Grade oligos rack up considerable saving and also facilitate speed in completion and delivery.

    For standard PCR and sequencing. Oligos below 30 bases are acceptable with PCR Grade. PCR and sequence can tolerate up to 50% truncated sequences. Anything above 30 bases, you are highly advised against using crude oligos for these applications.

    If sequencing target are large templates like BAC, cosmids or bacterial genimes, it is highly advised to have them TOP purified

  11. What is TOP purification and what can it be used for?

    TOP: Trityl-On Oligonucleotide Purification
    TOP is a simple, high-throughput approach to oligonucleotide purification. TOP provides greater than 90% pure full-length oligo and is ideal for applications that require high-quality DNA. It efficiently removes truncated failure sequences generated during the synthesis process that do not contain a 5' dimethoxytrityl group. In addition, deprotection solution salts and by-products (i.e. benzamide protecting groups) are also removed simultaneously.

    TOP chemistry is based on the principle of reverse-phase (RP) chromatography. Oligonucleotides purified by TOP are synthesized with the final 5' terminus protecting group [trityl or dimethoxyltrityl (DMT)] left on the oligo. The hydrophobic nature of the trityl group permits tighter retention of the desired full-length oligo than the truncated failure sequences that do not contain a trityl group. The failure sequences are washed from the tube with a low percent acetonitrile rinse. Retained oligonucleotides are then detritylated on column with trifluoroacetic acid (TFA) to remove the acid-labile trityl group. Residual acid is washed from the tube with two rinses. The full-length oligo is recovered in its purified form with an aqueous-organic solvent.

  12. Why is there no amplification with my primers?

    There are many reasons why PCRs don't work. To ensure success, please observe the following steps:

    • Vortex your Mg2+ stock or the 10X reaction buffer if it contains Mg2+. Frozen solutions with Mg2+ form a gradient in the tube when thawed. Vortex thawed reagent tubes thoroughly for at least two minutes to ensure proper mixing.
    • Use powder-free gloves when setting up reactions as glove powders inhibit thermostable polymerases.
    • Use primer design software to design PCR primers. Programs such as Primer 3.0 which is freely available through a web-interface can help design your PCR and primer sequence selection. Selecting and designing a sequence by hand is fraught with many pitfalls.
    • Adopt good laboratory practice to prevent carry-over contamination.

  13. What is carry-over contamination and how can I prevent it?

    Carry-over contamination is the most prevalent cause of amplification failures. It is not only the carry-over of previous positive PCRs but also primers or even other previous contaminating templates. This usually leads to false results.
    Controls are very important in any laboratory procedure. Always include positive AND negative controls along with any PCR you are attempting. A common plasmid can be used as a positive control template together with appropriate MCS flanking primers. Negative controls usually use water as the "template". Controls determine reagent fidelity.

    You can prevent carry-over contamination by:

    • Dividing areas for reagent mixing and sample preparation from areas for product analysis.
      This separation should be considered very seriously. No equipment or reagent which has been placed in the room for PCR product analysis should ever be transferred back to the room where the sample and PCR reagent are prepared. There must be specific equipment in both rooms. If these physical precautions are taken, most problems can be prevented.
    • Replacing ALL reagents and stock buffers with NEW chemicals and NEW water.
      Contamination may occur in more than one solution, or it can be in a stock solution where it may be difficult to find the source. The time spent in determining the contaminant exceeds the costs of exchanging all the reagents.
    • Using positive displacement pipettes or filter-protected pipette tips.

      Most laboratories might not have divided areas for PCR with reagent mixing, reaction setups and analysis. In this situation, pipettes are the main cause of most PCR failures.

      Researchers perform many PCRs and the contamination builds up in the shafts of pipettes usually leading to "no amplification" reactions. A positive displacement pipette will address this issue. If a positive displacement pipette is not viable, filter-protected pipette tips are the next best tool for prevention. Unlike positive displacement pipettes, filter tips cannot eliminate carry-over contamination completely, but do not dismiss them as a part of your good laboratory practice. There have been known cases where PCRs failing with pipettes using normal yellow tips and succeeding when filter tips are used.

  14. What quality control steps does 1st BASE perform?

    At 1st BASE, quality assurance is of utmost important to us. As such, we carry out our 5-step QC procedure on every oligo that leaves our laboratory:

    • Triple-step trityl monitoring during synthesis for every oligo
    • EVERY oligo is PAGE analyzed
    • Weekly batch sampling by RP-HPLC
    • Weekly in-house PCR tests
    • "QC Recall Program" - No oligo leaves our lab if pre-delivery QC requirements are not satisfied. For post-delivery QC, if we have the slightest reason to suspect quality is compromised in any way, our customer will be alerted and the replacement sent immediately with the highest priority.

    Should you face any problems with your oligos, please write to us with the Synthesis ID of your oligo and we will conduct our investigation. If there is any reason to suspect the quality of our primers, we will provide a free replacement immediately.

  15. Why is there no amplification with my primers?

    There are many reasons why PCR don't work To encusre success, please observer the folloeing step

    At 1st BASE we recognize and know that ALL Oligos manufactured will consist of a population with mutations. Mutations arise in some regions in Oligo sequences which, because of their folding properties, are very difficult to synthesize and therefore have an increased mutation rate at precisely these points.

  16. Why are there "mutations" in my oligo?

    Good question. This is what a lot of oligo companies don't want to talk about but we at 1st BASE have something to say about it.

    At 1st BASE we recognize and know that ALL Oligos manufactured will consist of a population with mutations. Mutations arise in some regions in Oligo sequences which, because of their folding properties, are very difficult to synthesize and therefore have an increased mutation rate at precisely these points.

    Other reasons are limitations in chemistry and synthesis technology. These are not mistakes during manual typing of sequences (sequences are imported electronically and error free into the production software), but the errors affect only a small portion of the molecules and are spread randomly over the sequence at a very low rate. We know that mutations are very frustrating for our customers, therefore we invest a lot of effort in our 5 step QC. Above this we also do periodic QC checks on our synthesizers to ensure they operate efficiently.

    The mutation rate is not a problem for methods like sequencing, hybridization, gel shift etc., because the mutation background is too low. Very rarely frustration may arise when Oligos are used for cloning projects because mutations from Oligos will become visible. In this case it is important to sequence further clones because there is a good chance that you will find the correct clone.


  17. What are these mutations exactly?

    Assuming a worst case scenario with an average coupling efficiency of 98.5%, a typical PCR grade 30 residue long oligo synthesis would have yielded around 50% full length products. Theoretically, if a PCR is performed and the PCR products cloned and sequenced, out of 100 clones, 50 will yield full-length sequences. The rest will be made of truncated oligos of various degrees ranging from 1 to 6 residue truncations. Picking a 6 residue truncation clone is rare but 1-3 residue truncations are common. There are also insertions and deletions seen in these cases though they are encountered less frequently than truncations.

    Assuming we take this same oligo and performed some sort of purification like PAGE, this will raise the number full length clones obtained from 50% to 85%. A rather comforting number but why is it not close to 99%? Other factors like cloning artefacts, PCR related secondary structures and even bacterial recombination events contribute to the loss of that 14%. As you can see, this is a numbers game. 1st Base recommends you pick at least 3-5 clones before reporting to us any wayward oligo. Re-synthesis followed by PCR and then cloning is a time consuming process. A lot can be saved if that next clone that you could have picked is correct.

    An interesting note about these aberrations is that they are distributed evenly throughout the length of the oligo. When analyzed by MALDI-TOF or PAGE, these aberrations are not even significantly visible!

    Therefore even with the highest purity purification, stringent QC using MALDI-TOF and CE, mutations should always be expected therefore temper your optimism by picking at least 3-5 clones.

  18. Will I eliminate mutations with purified Oligos?

    Additional purification is definitely recommended especially for Oligos used for cloning projects. However, the TOP-, PAGE- or HPLC-purified oligos (and the PCR products obtained with them) should be expected to give some mutant clones (clones with sequence infidelities, originating in the oligos). Purification reduces the amount of mutant Oligos but does not eliminate them.

    Inclusion of restriction sites on the 5' ends of the oligos to facilitate cloning PCR products can ensure fidelity. These enzymes will only recognize their specific palindromic sequences thus eliminating majority of the internally deleted (or addition) products. With the advent of TA cloning, most have forgotten that this can actually reduce mutant numbers by a large degree even without using purified Oligos.

    Keeping Oligos below to 35mers in length also keeps the mutations low because synthesis infidelities are increased as the oligo length is increased.

    In general, selection and sequence analysis of 3-5 independent clones is still advised (not just one or two clones).


  19. Can you make the oligos having a high percentage of 'G' residues?

    It is known that oligos having a high percentage of 'G' residues are difficult to synthesize, especially if the sequence contains a run of 'G'. It is also reported if there is a run of four or more 'G', oligos tend to aggregate and form "guanine tetraplex". (Poon and MacGregor, Biopolymers, 1998, 45, 427-434.) By substituting inosine for some of the 'G', the formation of "guanine tetraplex" can be disrupted.


  20. How many bases are needed before restriction sites on the 5'-ends of oligos

    Generally, 4-6 bp is a good length to protect your site with although this can vary to be as few as 1bp and as many as 9bp. Check with your enzyme catalog for info

  21. Why can't i clone double stranded Oligo directly?

    synthetic oligos do not have the 5' phosphate group necessary for ligase to work. To clone directly you must add a 5' phosphate using Polynucleotide Kinase(PNK) or order them nade with a chemical phophate added (at extra charge).

  22. What extras can I expect from 1st BASE?

    At 1st BASE, we believe in giving good value.
    We provide some little extras such:

    • Comprehensive oligo datasheet with detailed specifications of your primer order
    • Colour coded caps for easy identification
    • Saturday deliveries
    • Late ordering; order as late as 7pm for your orders to reach you the next week day (standard PCR grade primers)
    • Oligos can be delivered lyophilized or as a 100 µM stock solution, simply indicate on ordering
    • Lastly, you can count on us to have regular promotions and specials on our products and services, so that you can get a good deal!

  23. I need help with the design of siRNA duplexes or Dual-labeled Fluorogenic Probes, would 1st BASE be able to help me?

    1st BASE will provide complimentary hydrolysis probe design assistance for confirmed probe and primer sets orders. 1st BASE will also provide complimentary siRNA duplex design assistance for confirmed orders. See Products > DNA/RNA Synthesis > Ordering

    Simply write to us and include the Gene Accession number of your Gene of Interest. We will provide our proposals within 3 working days, upon your confirmation of purchase.

  24. What guarantees come with 1st BASE's proposed probe / siRNA

    We will synthesize siRNA or probe sequences based on sequences requested. Where required, we will provide design assistance. Typically we provide 2-3 proposals but our customers are required to BLAST and check the proposed sequences based on their application requirements. We use three different algorithms to select the siRNA sequences. We will be able to guarantee the quality and purity of our specialty products, and that at least 1 out of 3 proposed specialty products should yield positive results, so long as the necessary control reactions are set up.

 

Answers - Gene Synthesis

  1. What is the difference between custom oligos and a synthetic gene?

    A standard oligo synthesis will yield single stranded DNA bases. Typically oligo synthesis can yield up to 100mers in length. In synthetic gene synthesis, we will be able to produce a length of dsDNA cloned into a vector of choice. The length of the DNA fragment from gene synthesis can be much longer, between a couple of hundred bps to a few kbs in length.

  2. What Quality Control steps does 1st BASE include to ensure 100% fidelity in Gene Synthesis Projects?

    As part of the quality control process, we run a RE digest to select positive clones and submit these clones for DNA sequencing. The sequences yielded must be a 100% match back to the original sequences submitted. These data will be included when the project is completed and delivered to our customers.