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Welcome to our new Ask An Expert blog.  Answers to frequently asked questions  are listed by topic on the Ask An Expert homepage.  Please feel free to ask an expert by clicking on the comment link below. Thank you!

24 thoughts on “Ask An Expert!”

  1. Hi,
    I just want to know the standard protocol to link an acid to an amine group.Acid is in my complex, and the DNA I use has a C12NH2 linker at the 5′ end. I want to link my acid to this NH2 group in the linker. Thanks!

  2. Hi,

    Could you please let me know what is the standard protocol to link an acid to a DNA with a linker of C12NH2 at 5′ end. (I want to link C00H group of my complex to NH2 of the linker). Thanks!

  3. Thank You Anton, I would also like to know that how long the efficiency of phenol can resist, and how to know that the efficiency has lost?

    • Dear Supriya,

      We do not supply phenol, so we have not done any stability studies. The manufacturer may be able to provide stability information for you. I would recommend storing phenol at 4 deg C and replacing it if you see color in your pellets.
      Thank you,
      Anton

  4. Thank you so much. Well I ve some other issue too. I am working with some marine animals and Isolation of DNA is an issue. i am getting colored aqueous layer and that results a colored pellet too. I am not understanding why?

    • Hello Supriya,

      Sometimes the aqueous layer of phenol extractions (or Trizol extractions) can contain a pink color which precipitates with the pellet. For many applications this is not a problem. Often this can be solved by buying a new batch of phenol or Trizol.

      Thank you,
      Anton

  5. I have some sequences which is not matching with the primer even not with the 3 prime end but when i blast it, it shows similarity with the same species and i verified with it with taxonomic ally and morphological. So am sure that the product is been sequenced properly then why it is not matching with the primer.

  6. Hello Natasha,

    Thanks very much for the information and the references – it was all very helpful. Having read the papers, I think that the dPTP and 8-oxo-G approach seems the most suitable for my work. I was aware that these bases had been used in primers to introduce mutations, but I didn’t realize that they had been used as dNTPs.

    I would certainly also be interested in hearing more about your mutagenesis product line.

    Thanks again,

    Daniel

  7. Hello there,

    Are there dNTPs that I can use in a PCR to induce random mutations? I’m imagining mixture of the normal 4 dNTPs supplemented with a small proportion of some that are less choosy about which bases they hydrogen bond to. After a few rounds of PCR, I’d clean the products up and would then reamplify using just the 4 regular dNTPs.

    Thanks for any help you can give,

    Daniel

    • Hi Daniel,

      Thank so much for your question. There are a number of dNTP analogs which have been reported to introduce mutations in PCR. One of the most well-studied set of analogs includes dPTP (cat # N-2037) and 8-oxo-dGTP (cat # N-2034). When used in equimolar concentration to dATP, dCTP, dGTP, and dTTP, a number of transition and transversion mutations can be introduced.
      For further experimental details on the use of these analogs in PCR, please refer to the following references from Zaccolo et al.:

      – J. Mol. Biol. (1999) 285, 775-783.
      – J. Mol. Biol. (1996) 255, 589-603.

      Other mutagenic dNTPs which have been used in PCR mutagenesis schemes
      include:
      – dITP (cat # N-2012): Nucleic Acids Res. (1993) 21, 777-778.
      – 5-bromo-dUTP (cat # N-2008): Mol. Biol. Rep. (2008) 35, 663-667.
      – 2-hydroxy-dATP: Biol. Pharm. Bull. (2004) 27, 621-623.

      Your strategy for the use of the modified dNTPs in a first PCR to introduce the mutations, followed by a second PCR using the natural dNTPs is precisely how PCR random mutagenesis schemes are performed. For each of the analogs which have been described, the ratio of modified dNTPs relative to the natural dNTPs was optimized. Therefore, I would refer the primary references listed above for the experimental conditions that are specific for a given dNTP analog.

      On a related note, TriLink is actively developing a mutagenesis product line that employs modified dNTPs. We anticipate launching this product in 2011 and can keep you up to date on these developments if you would like.

      Please do not hesitate to let us know if you have any further questions.
      Best of luck with your experiments!
      – Natasha

  8. When requesting an oligo with multiple couplings of the same internal modifcation your ordering system gives out a note that the additional couplings will be discounted upon order. What does it actually mean? Does it mean we are only charge for the first modification?…..For example a 60-mer DNA oligo having a total of 5 /5-Bromo-2-deoxyuridine/ modifications

    • Dear Steve,

      For many of our modified bases, a discount is extended when multiple incorporations of a particular base are required within a single oligo. After the first incorporation a 25% discount is placed on the second incorporation and a 50% discount on each additional incorporation. If you would like to request a quote click here.

      Best regards,
      Judy

  9. Hi,

    I am looking for a strategy to use click chemistry to label RNA in cells infected with an RNA virus. We wanted to use a strategy of using ethynyl uridine labeling in the presence of actinomycin D to watch the location of viral RNA in real time after the addition of the azido-fluorescent probe. The only problem with this is that the high cost of the ethynyl uridine is somewhat prohibitive for our experiments. Do you have any suggestions? Do you make 2′ alkynyl nucleoside triphosphates? If not, would you know who might be able to help me?

    • Dear Jason,

      Thank you very much for your interest in modified nucleoside triphosphates which are amenable for use in Click chemistry RNA labeling schemes. Although we do not carry 2′-alkynyl nucleoside triphosphates, we carry several azido-functionalized analogs which may work for your application, such as 2′-azido nucleoside triphosphates (cat #: N-1028, N-1029, N-1045, and N-1063). For incorporation of 2′-modified NTP analogs during in vitro transcription, we recommend T7 R&DNA™ Polymerase from Epicentre.

      While the use of azido-functionalized nucleoside triphosphates will not allow for Click chemistry with an azido-fluorescent probe, a simple change to a fluorescent probe with a terminal alkyne should work. Click here to request a custom oligonucleotide quotation.

      I hope this helps – do not hesitate to let me know if you have any follow-up questions.

      Good luck!
      Natasha

  10. Pingback: Long Oligonucleotides « Ask An Expert

  11. Hi,

    I am searching for a way to obtain oligonucleotides longer than 100bases e.g 200, 300 … upto 500bases.

    I know that synthesis of oligos >100bases becomes difficult. But is there some way to generate 100base fragments and then chemically ligate them? Does Trilink offer this type of service ?

    I am not very particular about the sequence of the oligos (polyA or polyT will do) or the exact length (e.e 205 instead of 200 will be fine).

    Please feel free to contact me directly by e-mail: ppal _at_electronicbio_dot_com

    Thank you.

    Prithwish

    • Dear Prithwish,

      Chemical ligation is still not the best way to accomplish what you wish to do. Either linkers will exist between the fragments in some cases, or efficiency will be low. Enzymatic ligation is still the best way to go.

      Order 100mer fragments, and then order shorter fragments (30 – 40 will suffice) that are complimentary to, and bridge across the fragments. The fragments to be joined require a 5′ phosphate which is a reasonably simple modification to an oligo. Then ligase is used to join the fragments together.

      Although this is not a service we normally provide, we will be happy to discuss this with you further if you would like a quotation for this work. Please contact our team at sales@trilinkbiotech.com and refer to this blog question.

      I hope this helped. I will be happy to provide more details.

      Best regards,
      Rick

  12. Hello

    Can you recommend a commercially available Taq poly.. that works best at incorporating Biotin-16-Aminoallyl-2′-dCTP (N-5002)?

    Thanks

    Al

  13. Hi, Richard,

    I am quite new to oligonucleotide chemistrty. For my research project, I would like to link an DNA molecule to an RNA molecule. The following is the product I would like to produce:
    5′-(GGT…TGG) -linker-(UAG…GUU)-3′.

    Currently I have both oligonucelotides and ribonucleotides. I am looking for a linker that can link these two together. Or I am wondering if you can help me synthesis this product.

    I am looking forward to hearing from you. Thank you very much!

    Yun Wu

    • Hi Yun!

      This is a good question and one we answer frequently.

      The conjugation of two oligonucleotides together is not readily accomplished after the compounds are made. In essence, you are seeking a chemical ligation. The problem is that you would be attempting to conduct chemistry on two of the less reactive sites on the oligo – the 5′ and 3′ hydroxyls. The exocyclic amines are much more reactive. Nature activates the hydroxyls using triphoshates and then an enzyme to distort the bond angles making the reaction possible.

      There is a way to react with the hydroxyls and not the amines. Silyl chemistry is hydroxyl specific. However, silyl linkers are not described for the application you seek.

      Our standard advice in this case is to resynthesize the strands with the end goal in mind. We would add the necessary chemical modification (a primary amine or thiol) that would allow us to use standard conjugation chemistry.

      In general, resynthesis of the strands with the appropriate modification is less expensive than trying to find a way to link two completed oligonucleotides.

      We are also capable of synthesizing the DNA-RNA sequence as one molecule. If you require a spacer, we have a number of different options that will offer a choice of linker length that could be included in the synthesis.

      I hope this helped. I wish there was a direct solution to your problem. On the other hand, we do know how to accomplish your overall goal of making a DNA-RNA chimera.

      Click here to view our spacer options.
      Click here to request a quote for your oligonucleotide.

      Best Regards,
      Rick

  14. Welcome to Trilink’s Ask An Expert Blog page. We are here to help in any way we can. Please feel free to leave a comment or ask a question about any aspect of oligonucleotide chemistry or nucleic acids in general.

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