Author Archives: TriLink BioTech

What are your recommended conditions for running a gel for mRNA or long RNA transcripts?

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The conditions we recommend for running a gel are:
We run a 1% agarose gel. Depending on the size of the gel/number of wells, we load 250 ng to 1 ug of mRNA. We dilute mRNA to 0.2 ug/uL, then add equal volume of NorthernMax Gly sample loading dye.

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For making a 1% agarose gel, we would use 50 mL of gel and 0.5 grams of agarose. Microwave the agarose until melted and allow to cool for ~10 min. Add 0.5 uL of ethidium bromide. Swirl gently to mix. Pour gel.

What’s the difference between the CleanTag™ Ligation Kit and CleanTag™ Library Prep Kit?

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Both products included the reagents needed for the ligation of the 3′ adapter and 5 ‘ adapter. The CleanTag™ Small RNA Library Prep Kit (Cat# L-3206) also includes the reagents needed for the RT and PCR steps of library preparation which were not included in the CleanTag™ Ligation Kit (Cat# L-3203).

CleanTag™ Small RNA Library Prep Kit
(Cat# L-3206)		 CleanTag™ Ligation Kit
(Cat# L-3203)
CleanTag™ 3′ Adapter CleanTag™ 3′ Adapter
CleanTag™ 5′ Adapter CleanTag™ 5′ Adapter
Enzymes, 1 & 2 Enzymes, 1 & 2
Buffers, 1 & 2 Buffers, 1 & 2
Reverse Transcriptase
RT Buffer
dNTP Mix (10 mM ea)
DTT
High Fidelity PCR Master Mix (2X)
RNase Inhibitor RNase Inhibitor

The CleanTag™ Ligation Kit was discontinued in early 2016.

Which dyes are available and are there specific dyes that are most compatible with active translation? Is there one specific nucleotide that can accommodate the dye better than others with respect to translation?

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Bulky dyes may block the ribosome during translation and we have seen lower translational efficiencies with more substitutions. We recommend not to fully substitute, but only substitute 10-50%. For our catalog products, we use a 25% substitution of the dye-labeled nucleotide. Please contact us for any additional information.

Are 6-FAM-labeled oligonucleotides freely taken by mammalian cells in culture in the absence of transfection agent ? (e.g. a liposome). In other words is 6-FAM permeable to the cells?-Carlos

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Dear Carlos,
6-FAM isn’t hydrophobic enough to difuse an oligo across the cell membrane. Some modifications that help with cell uptake are multiple incorporation’s of spermine, or hydrophobic labels like stearyl and cholesterol. Please contact us if you have any additional questions.

Kindest regards,

Danica

How much mRNA is needed to transfect cell cultures in 24-well plates?

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There are typically two avenues for transfection of mRNA depending on cell type:

1. For adherent cells, use a transfection reagent such as MessengerMax (Invitrogen), mRNA TransIT (Mirus) or mRNA-In (MTI-GlobalStem).

a. For a 24-well plate format, use a ratio of 500 ng mRNA:1 ul transfection reagent in a total volume of 50 ul complexed mRNA/lipid per well. This is a good starting point and is described in this protocol. Diluting your working stock of mRNA down to 100 ng/ul works well to establish manageable volumes for mastermixes.

b. We’ve only used TransIT and mRNA-In in-house – from our experience these reagents give a dose response of FLuc activity from between 100 ng to 400 ng. Therefore, a minimum starting amount of 100 ng is sufficient. Use 100 ng mRNA: 1ul reagent in a complexed final volume of 50 ul per well.

c. Ratios for other plate formats need to be optimized according to the manufacturer’s instructions.

2. For cells in suspension, such as CD34+ cells, electroporation has traditionally been the mode of mRNA delivery. However, transfection reagents have advanced and some are able to transfect cells in suspension reliably, such as the transfection reagent mRNA-In (MTI-GlobalStem).

a. For electroporation, an instrument from Lonza is recommended based on advice from a trusted collaborator.

b. Use 1-15 ug of mRNA per million cells in a 100 ul volume.

Hello, We would like to use CleanAmp™ dNTPs in our PCR but were unsure if they will de-protect efficiently at 92°C since that’s the temperature we’ll doing denaturation initially and at every cycle. -Thank you, Chun-Nan

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Dear Chun-Nan,
The CleanAmp™ dNTPs have a half life of 5 minutes at 95°C in PCR buffer. At 92°C the deprotection rate will be slightly slower however this temperature should allow deprotection of enough of the CleanAmp™ dNTPs to work in your PCR. If needed, you could extend the time of the initial denaturation. Please contact us if you have any additional questions.

Best Regards,
Sabrina

What protocol should I use for labeling Aminoallyl FLuc mRNA?

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Suggested protocol for labeling Aminoallyl FLuc mRNA

This protocol is compatible for use with most water-soluble NHS esters*. If using a non-water soluble NHS-ester, different processing will be required. Precipitation in alcohol, such as isopropanol or ethanol, should be considered to remove excess NHS ester and any hydrolysis products. Multiple precipitations may be required.

*This protocol has been tested at small scales (100 ug) using Aminoallyl FLuc mRNA and Cyanine 5. Scales larger than 10 mg may require modifications to the protocol. The protocol has been designed to minimize mRNA degradation during the labeling process.

Salt exchange of Aminoallyl FLuc mRNA
1. Concentrate desired amount of Aminoallyl FLuc mRNA with 3K centrifugal filter. Reduce volume to ~0.25X of original.
2. Add 100 mM NaHCO3 to achieve total volume of 1.25X of original.
3. Repeat steps 1-2 at least three times. Add enough 100 mM NaHCO3 at final step to achieve 1X original volume of product. Concentration should be at ~ 1 mg/mL.

Labeling Reaction
4. Add 1X volume 100 mM NaHCO3 in H20. Mix gently by inverting several times.
5. Add 2X volume 2 mM NHS ester solution in DMSO. Mix gently by inverting several times.
6. Incubate 90 min at room temperature. If label is fluorescent, make sure to shield from light.
7. Add ~0. 35X volume 4M hydroxylamine in H20. Mix gently by inverting several times.
8. Incubate in dark for 15 min at room temperature. If label is fluorescent, make sure to shield from light.

Processing
9. Concentrate ~3X with 10K centrifugal filter device.
10. Add back 3X volume H2O.
11. Repeat steps 9-10 at least 6 times.

We recently purchased eGFP mRNAs (L-6101 and L-6402) as controls for mRNA delivery experiments. I have transfected HEK293 and DU145 cells using lipofectamine 3000 and PEI (5:1 weight ratio) and am unable to see GFP expression (by FACS) after 7 or 24 hours. By FACS (looking at cy5) the delivery looks good. I have tried both the labeled (L-6402) and unlabeled (L-6101) side by side and transfected 100 or 200 ng/well (96-well plate) into cells at about 50% confluency. Control transfections using these cell lines with a CMV-eGFP plasmid produce almost off-scale GFP expression when measured by FACS. Is there something wrong with my protocol and what is the timing and level of GFP expression I should expect in comparison to a plasmid transfection?

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Dear Scott,

Thank you for your inquiry. I would suggest checking for mRNA degradation. Make sure you are using serum free reagents (ie Optimem) that are rigorously RNAse free. We use special pipets for mRNA and for example do not do minipreps or maxipreps with these pipets. RNasezap can be used to clean work surfaces and pipets. FACS signal from the Cy labeled RNA does not ensure that there was good delivery since the RNA could simply be trapped in an endosome. This experiment should work well in the 293 cells, we have no experience with the other cell line. Additionally, you make want to experiment with the timing. Though I would expect to see EGFP expression at your indicated time points, many factors influence expression and half-life. We and others typically see peak expression between 12-18 hours.

Regards,
Brea

Dear sir/madam : I am planing to do selex round. If I have ssDNA 25 random nucleiotids flanking by to primer biding site so totally 66mers. My targets protein will be MLL1. So I am wondering what are the factors to be considered regarding binding ratio of initial ssDNA library with protein and subsequent rounds? What should be the recommended binding ratio for the first and the following selex round?-Khun

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Dear Khun,

This is something you should determine experimentally for your protein of interest and your library.Our biologists have provided a few publications which describes a RNA and DNA selection protocol which may have additional information

Design, Synthesis, and AmpliÞcation of UNIT 9.2 DNA Pools for In Vitro SelectionIn Vitro Selection of RNA Aptamers to a UNIT 24.3 Protein Target by Filter Immobilization

Best Regards,

Tiffany Teng

Product Specialist I

If 10ul RNA was used for 3′ adapter ligation, would you suggest using 10ul in 5′ adapter ligation reaction or the whole volume (20ul) from 1st reaction would work too?-Anastasia

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Dear Anastasia,

Thank you for your email. While we recommend sticking to 2 uL of RNA input when possible, we found that we can still obtain good library prep results with up to 10 uL of RNA (without changing any other volumes in the 3′ Adapter Ligation Reaction). We recommend that you carry the entire volume of this reaction (10 uL + excess) to each of the next steps. This means that each subsequent reaction will now contain larger volumes than outlined in the manual.

Please let me know if you have any additional questions.

Regards,

Brea

What’s your guaranteed minimum yield (OD) for oligos ~60bp in length after HPLC purification on a 200nmol and a 1umol scale? -Ben

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Dear Ben,

At the smaller scales, 15 umole and below, there is no guarantee of final yield. Each oligo has unique synthetic properties based on its sequence and modifications; therefore it is not easy to predict how much material will result from a particular synthesis.

Our general expectations for HPLC purified unmodified DNA oligonucleotides are as follows:

0.2 µmole scale 5 – 15 OD260 units (˜0.15 – 0.5 mg)
1.0 µmole scale 20 – 60 OD260 units (˜0.66 – 2 mg)

If you require a specific yield, please let us know when you place your order or request a quote.

Regards,

Danica

I want to conduct 3D PCR as desribed by Suspene et al. I have ordered DIT and dDTP from your company. Is 2-amino-2′-dATP the dDTP that Suspene is describing? And is Pfu polymerase able to incorporate these nucleotides? -Bernadette

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Dear Bernadette,

Thank you for your inquiry. Yes, 2-amino-2′-dATP is the dDTP that Suspene describes. We do not know for sure whether pfu polymerase is able to incorporate these nucleotides but it probably doesn’t. We recommend trying NEB’s Therminator.

Please let me know if you have any additional questions.

Regards,
Brea

What concentrations of mRNA are considered high,100ng/ul? Which buffer should I use to store mRNA? My mRNA activity still drops after 3 months at -80C and stored at 100ng/ul. I’ll appreciate any help-Marcia

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Dear Marcia,

In general, 100 ng/uL is not considered a high concentration. We supply our mRNA at 1 mg/mL in 10 mM Tris-HCl, pH 7.5. That said, the mRNA sequence and structure can dictate its solubility. We recommend heating the mRNA for 15 min at 37°C to improve solubility. Long incubations at elevated temperatures should be avoided.

In regards to the diminishing activity of your mRNA, have you checked for degradation?  In addition to the solubility, your issues with mRNA activity may also result from RNA degradation over time. To combat degradation you should use RNase-free reagents and materials and use proper technique. Additionally, we suggest that you aliquot your RNA to limit freeze/thaw cycles.  A higher concentration may also improve stability however  could exacerbate your issues with solubility.

Please let us know if we can help you further.

Regards,

Brea

Hi, can Trilink synthesize a RNA di-nucleotide containing at least one intrisically fluorescent ribo-nucleotide analogue? Thank you-Egor

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Dear Egor,

We are able to synthesize an RNA dinucleotide containing an intrinsically fluorescent ribo-nucleotide analog.

I have included a link to the product page for a few options we can offer:

Isomorphic-Intrinsically Fluorescent

For a formal quotation, please contact us through sales@trilinkbiotech.com

Best Regards,

Tiffany Teng

Product Specialist I

What are the benefits of using mRNA over plasmid or viral-based approaches for vaccine and immunotherapy development?

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mRNA offers several advantages over traditional plasmid and viral-based approaches:

  1. mRNA boasts a superior safety profile. As a transient carrier of genetic information, it is metabolized naturally and poses little to no risk of genomic integration. Additionally, no inactivated viruses or pathogens are needed.
  2. mRNA serves the dual purpose of expressing the desired antigen as well as acting as an adjuvant.
  3. mRNA triggers a more diverse immune response. Because the mRNA encoded epitopes are intracellular, they are recognized by the immune system in an MHC class-independent manner.
  4. mRNA can more readily transfect difficult-to-transfect cell types because it functions in the cytoplasm. DNA vaccines can be limited by lack of access to the nucleus.
  5. mRNA manufacturing is easily scalable. Because mRNA transcription is carried out completely in vitro, to hundreds of millions of vaccine doses with a lead time of as little as a few weeks. This allows for rapid deployment of a new antigen during pandemics.
  6. mRNA is easily customizable. The ease of manufacturing makes it a viable option for personalized treatments.

I’m buying a ssDNA library from TriLink and doing SELEX for one target. Assuming I get one specific sequence that will bind with high specificity and affinity. My question is, can I patent that sequence, start conducting a clinical trial and then sell it without permission from TriLink?

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Dear Alan,

TriLink does not take any claim on an aptamer sequence determined from one of our Nucleic Acid Libraries for Aptamer Selection. Please let us know if you need any additional information.

Best regards,

Joey

Which “High Fidelity PCR Master Mix” would you recommend using with the CleanTag Ligation Kit for Small RNA Library Prep? I noticed your protocol uses more than the volume that comes with the TruSeq Small RNA kit, so I was wondering which PCR MasterMix have you found most success with? -Steve

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Hi Steve,

Thank you for your question. We recommend the Q5® High Fidelity 2x Master Mix (catalog # M0492S) from NEB for use with the CleanTag™ Ligation Kit for Small RNA Library Prep. You can view our full list of recommended reagents here.

Please let me know if you have any other questions.

Best regards,
Elizabeth

Can you ship RNA oligonucleotides (10nt) to New Zealand? If yes what will the shipping costs be? -Jack

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Dear Jack,

Thanks for contacting us. We can ship lyophilized RNA oligos to New Zealand by FedEx International Priority. The estimated freight cost to New Zealand is $65. The freight cost is based on FedEx daily rates and can vary depending on the shipping address. Please note FedEx does not allow dry ice shipments to New Zealand, so any product requiring dry ice would need to be shipped through a specialized courier coordinated by you.

You can request a quote for your 10 nt RNA oligo in OligoBuilder®. Please let me know if there’s anything else I can help you with.

Best regards,
Kaitlin

Is there an advantage in using a c12 linker over c6 linker in coupling probes to magplex magnetic beads from Luminex. The objective is to develop a 34 target multiplex assay for detection of various viruses and bacteria using the MagPix instrument from Luminex. -Rachel

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Dear Rachel,

Thank you for your question. We suggest using the C12-amino linker as recommended in the Luminex probe/primer design manual. It is likely that the protocol was optimized for this linker.

Please let us know if you have any additional questions.

Regards,
Brea

I just used your CleanTag Ligation Kit for Small RNA and I am trying to determine if the ligation/library prep was successful. Am I correct in assuming that I expect to see essentially no adaptor dimer formation in the prep? The TapeStation results show large (~2-4 ng/µl) peaks at around ~140bp for each sample (magnetic bead cleaned) and no obvious peaks at what would be the adaptor-dimer (~120bp). I just wanted to confirm that because of the CleanTag chemistry, no adaptor-dimers form during ligation, and therefore, they aren’t amplified. Is this correct? If true, I should also be able to load these preps straight onto a sequencer with no gel purification of the 140bp band? – Steve

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Dear Steve,

You are correct in that the CleanTag™ chemistry can fully suppress adapter dimer formation. If you are using very low input levels (less than 10 ng) you may start to see some adapter dimer but it should be minimal compared to amount of tagged library. Dilution of the adapters per the product insert is key to keep adapter dimer low at lower inputs.

Yes, you can load magnetic bead purified samples without gel purification directly onto a sequencer and get good quality sequencing data.

Best regards,
Sabrina Shore, MS
Scientist

I am interested in an aptamer that would be able to detect a Salmonella typhimurium cell at one end and able to specifically hybridize to a ssDNA at the other end. Is it possible for you, and how difficult would that be for such an aptamer to achieve and maintain its 3D conformation in vitro? – Aristea

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Dear Aristea,

Thank you for your interest in TriLink BioTechnologies, Inc. Unfortunately, we do not offer aptamer design or selection services. We recommend Base Pair Bio who should be able to help you with your research needs.

We can synthesize the aptamer once the sequence is determined. Please let us know once you have a sequence identified and we will gladly provide a quotation.

Thanks,
Kaitlin

I would like to order 3′-Deoxyadenosine-5′-Triphosphate and 3′-Deoxyguanosine-5′-Triphosphate (5 umoles each) and I’m wondering how much it will cost to ship to Cambridge, UK? – Gillian

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Dear Gillian,

Thank you for your inquiry. 3′-dATP and 3′-dGTP are shipped FedEx Priority International on dry ice. The average cost to ship to Cambridge, UK is $130 USD. Please note that our online cart uses general estimates based on country which can vary from the actual shipping cost. For example, since other regions in the UK can be more expensive, the online estimate is $140 but the final invoice will only reflect the actual cost of shipment, not the full estimate.

Best regards,
Elizabeth

I would like to block ligation at the 3′ end of an oligo. The traditional way to block described in the literature is C3 spacer. I was wondering if I label the 3′ end of an oligo with the Cy5 dye, would the dye molecule effectively block ligation? -Chun-Nan

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Dear Chun-Nan,

The blocking groups most often described in the literature are 3′ dideoxy-C, 3′ C3 Spacer (C3-OH) or 3′ Amino Linker (C6-NH2). All the mentioned modifications permanently block the 3′ hydroxyl group of the 3′ base. We can use the 3′ Amino Linker and label it with the Cyanine 5 dye which would continue to block the 3′ hydroxyl group.

Please contact us if you have any other questions.

Best regards,
Sabrina Shore, MS
Scientist, Research & Development

I obtained a oligo from Trilink 47 bp in length with the presence of a random fragment that was 10bp. Scale ordered: 0.2umole Extinction coefficient: 457.1 units/umol Od= 63.5 MW= 14334.9 g/mole I calculated the molarity as umol= OD/extinction coefficieint = 63.5/457.1= 0.138umol To make 1mM of the oligo I added 138ul of water to the mixture. Now total MW= 0.138 * 14334.9 = 1978.2ug But as there are 138 ul each ul should have a concentration of 14.339 ug but each ul of my sample had 1 ug concentration. Is there an error to my calculations? -Anjali

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Dear Anjali,

Thank you for your inquiry. A final concentration of 1 uM means you have 1 μmole or 14.34 ug per L. Concentrations should be confirmed with OD readings of the final solution. Please remember, the extinction coefficient and molecular weight reported is calculated average and the final values of a randomer oligonucleotide may vary.

We’re happy to discuss how to measure the concentration of you solution. Please email us to discuss measuring the absorbance.

Best regards,
Elizabeth

I want to add carboxyl-dCTP to the DNA fragments after RE digestion with Klenow. However, I saw that “5-Carboxy-dCTP is unstable in neutral and acidic conditions.” I wonder if 5-Carboxy-dCTP is stable in NEB buffer 2 (pH=7.9), or what is the best condition to do this. -Venson

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Dear Venson,

Thank you for your inquiry about 5-Carboxy-dCTP. This product was functionally tested when it was added to our catalog.

In a single pase pair extension assay, 5-Carboxy-dCTP incorporated less efficiently than dCTP with Klenow using a buffer of pH 7.9. 5-Carboxy-dCTP was also tested in PCR amplification of a ~500 base pair Lambda gDNA in a pH 8.4 buffer. The PCR product formed with 75% substition of dCTP. Our chemists believe that 5-carboxy-dCTP should be stable enough in NEB buffer (pH 7.9) to be used with Klenow DNA polymerase but please remember to store the product at -20°C.

Please let me know if this provides adequate information for your experiments.

Best regards,
Elizabeth

I’m working with magnetic nanoparticles (10 nm) for hyperthermia approaches and I want to functionalize (coat) it with DNA (aptamer). what is the best conjugation method that could be used? – Mahmoud

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Dear Mahmoud,

Thank you for your inquiry. TriLink is able to offer different attachment chemistries. Based upon your application, we would match up the functional group on the oligo based upon the reactive group of the magnetic nanoparticles. Some possible chemistries include Thiol/Maleimide or NHS-ester/primary amine of a linker.

TriLink’s DADE (decanoic acid diester) linker offers a novel way of preparing conjugates more economically and with much more flexibility. We can readily prepare 5′ carboxyl linkers. This linker can be used to conjugate to amines using conventional carbodiimide chemistry. Though less convenient than the solid phase method described in DADE: A Pre-activated Carboxyl Linker, Applications and Methods, solution phase methods may be necessary at times. The DADE linker is also useful for conjugation to amine bearing molecules.

We can also employ other attachment chemistries if you can provide the reactive group on the nanoparticles. Please contact our Product Management group to continue the discussion.

Best Regards,
Katelyn

I’m interested in the 2’OMe modified nucleotides (N-1015-18) and also in 2’F and 1-Thio (N-1007 and N-8005). Have you tested if these modifications can be incorporated by transcription in vitro using T7 RNA Polymerase? if yes, in what efficiency? -Moran

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Dear Moran,

Thank you for your question. We see a modest amount of incorporation of 1-Thio-ATP (N-8005) using T7 polymerase (≤25%). However, preliminary studies have shown greater incorporation the other 1-Thio analogs (≥50%). For 2´F incorporation (N-1007) we recommend using T7 R&DNA™. Though we have not tested 2´ OMe analogs with wt T7 polymerase, this paper suggests that T7 polymerase with a Y639F/H784A double mutation is optimal for incorporation.

Please let me know if you have further questions.

Regards,
Brea

I have ordered 5’end-modified RNA oligonucleotides and would now like to 3’end label them. Unfortunately, I cannot find any info on the nature of the 3’end of your RNA oligos. Is it a standard -OH group? -Eva

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Dear Eva,

Thank you for your interested in our RNA oligonucleotides. You are correct, the 3′ end of an unmodified oligonucleotide would be the terminal base in your sequence with hydroxyl groups (-OH) on the 2′ and 3′ sugars.

We also offer a variety of linkers that can be used to have different chemistries on the 5′ and 3′ end of an oligonucleotide. You can use an amino linker for reactions with and activated carbonyl and a thiol linker for reactions with maleimides. The order of the chemistry performed is important as a maleimide will react with the free amine.

Please let us know if you’d like to discuss your project or quote a new oligonucleotide.

Best regards,
Elizabeth

I have been reading about aptamers, with the hope of working with them in our lab soon. As I understand, the three dimentional conformation taken up by an aptamer on binding to its target remains the same, through every batch of that aptamer synthesized. I could not however find an article about this. Could you help me with this please? -Swan

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Dear Swan,

Thank you for your questions regarding aptamer conformation. Aptamer conformation is not straightforward, as a given sequence can take on multiple conformations. While some aptamers maintain one primary conformation, others do not. The secondary structure can depend on the actual sequence of the apatmer as well as external factors, including the pH and composition of the dilution buffer. In regards to whether aptamers can behave differently due to batch to batch variations, one can imagine a scenario where purity may influence structure and function. However, in general the synthesis should not affect aptamer conformation. Interestingly, we too were not able to easily find any online literature that specifically documents this phenomenon, though The Aptamer Handbook, edited by Sven Klussman presents a lot of useful information on aptamer activity, selection and design.

Please let me know if I can help you further.

Regards,
Brea

How can I design the best flank region with software? – Parisa

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Dear Parisa,

Thank you for your question regarding SELEX design. In his 2009 paper titled Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection, Dr. Andrew Ellington suggests using primers of approximately 20 nucleotides due to their melting temperature. To avoid secondary structure formation, he suggests using MIT’s web based program PRIMER3. Please let me know if you have further questions.

Best regards,
Elizabeth

I can’t do my RT reaction at higher temperatures. Will I be able to amplify challenging RNA targets with the CleanAmp™ One-step RT-PCR Master Mix?

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The CleanAmp™ One-step RT-PCR Master Mix provides a Hot Start RT step, which prevents amplification of unwanted side reactions and truncated products due to RNA secondary structure. At 47°C, a small amount of denatured RNA template will be present to generate just enough of the correct cDNA to go into the PCR reaction. A key step in developing the CleanAmp™ One-step RT-PCR Master Mix was ensuring amplification of targets with high secondary structure, such as ABCA7 and PBGD.

In order to obtain accurate concentration measurements using extinction coefficients provided by Trilink Biotech, what variables need to be held constant? I am assuming that extinction coefficients are determined experimentally by Beer’s law, when volume and path length are held constant. Can you provide details? Thank you, Jacob

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Dear Jacob,

Thank you for your question. TriLink calculates the extinction coefficient of an oligonucleotide by using the nearest neighbor method and reports it on the Certificate of Analysis. You can learn more about extinction coefficients in our Technical Article, An Introduction to Extinction Coefficients and Molecular Weights of Oligonucleotides. After diluting an oligonucleotide, the concentration can be determined by measuring absorbance with a spectrophotometer with UV lamp and quartz cuvette. Beer’s law can be used to calculate the concentration. If your oligonucleotide was supplied lyophilized, I recommend diluting to your preferred concentration using the method described.

Please let us know if you have any further questions.

Best regards,
Elizabeth

Do you know if the aptamers of the SELEX library can distinguish between two peptides with a single aa change, between Gly and Pro. Is there any way compare the likelihood of a certain site to fit such recongnition? Is it different between 20/30/40 nt libraries? Thanks- Eyal

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Dear Eyal,
Thank you for your interest in TriLink’s Aptamer Libraries. The complexity of the final library varies with the random region length. Libraries with longer random regions have more unique sequence motifs than libraries with shorter random regions. However, not all possible unique sequences can be represented in each selection. In contrast, libraries with shorter random regions will give you a better representation of all possible sequences but are inherently less complex than a library with a longer randomer region. However, once an aptamer is selected shorter aptamers are easier and less expensive to synthesize

Aptamers have been designed to bind to specific amino acids.Geiger et. al. found an aptamer that distinguished with a 12,000-fold improvement between L-arginine and D-arginine. I don’t know the specific discrimination between Proline and Glycine. It would need to be determined experimentally. Proline and Glycine have different structures but the location within the protein and the protein folding may affect the ability to find an appropriate aptamer.

Good luck with your experiments.

Best regards,
Sabrina Shore, MS

I would like to synthesize an mRNA that contains a modified nucleotide that I have prepared. I would like to place this nucleotide as a specific site of incorporation and have prepared a phosphoramidite version. Can you custom synthesize something like this? -Christopher

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Dear Christopher,

Thank you for your interest in TriLink. We have the ability to use custom phosphoramidites for oligonucleotide synthesis and routinely do so. Depending on the final length of your construct, we also offer chemical addition of a 5′ N7-Methyl-G cap to mimic natural mRNA.

We will need to discuss your project to understand the full request and discuss the options to incorporation a site specific mRNA modification. Our Product Management will be in contact with you shortly.

Best regards,
Elizabeth

How do I concentrate mRNA?

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mRNA can be concentrated using an Amicon® Ultra 30 kDa or 100 kDa size exclusion filter. These are available from EMD Millipore in various sizes. Simply add the mRNA to the filter and spin for a short time (~3-5 min) at the recommended speed. mRNA will be retained in the top chamber. Spin in brief rounds of centrifugation until the desired concentration is reached.  Note that over-concentrating the mRNA could lead to precipitation. Carefully remove the mRNA from the top chamber using a pipette. Calculate the actual concentration of the mRNA using a spectrophotometer or a Nanodrop™ device.

 

I would like to request quotation for a thiol-modified oligonucleotides. Is it possible to cross link the thiol-modified oligos to gold surface with succinimidyl 4-[maleimidophenyl]butyrate (SMPB)? What is the differences between 3′ C3, 3′ C6 and 3′ C6 Disulfide linker? -Lim

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Dear Lim,

Thank you for your interest in TriLink’s custom oligo synthesis. Thethree thiol linkers you mention differ by the position on the oligonucleotide (5′ or 3′) and the linker length (3 or 6 carbon chain). The structure of the reduced thiol linker can be found on each product page. Thiol modified oligonucleotides are shipped as the protected thiol (for example, C6-S-S-C6, aka disulfide) to avoid dimerization. Prior to use, the disulfide can be reduced using TCEP. Thiol modified oligos can be ordered through OligoBuilder®.

Thiol modified oligos can be direclty linked to a gold surface as described by Li Z, Jin R, Mirkin CA, Letsinger RL. .

Please let us know if you have any additional questions.

Best regards,
Elizabeth

We are interested in ordering 5′ Maleimide-modified oligonucleotides to label them with a thiol-reactive reagent for RnD assays. If we supply the thiol-reactive reagent, do you are able to deprotect the maleimide, to make the conjugation with our reagent and to purify the conjugated product? – Michael

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Dear Michael,

Thank you for your interest in TriLink and the 5′ Maleimide-modifier. TriLink specializes in working closely with our customers for the individual requirements of each project including working with customer supplied material for various types of reactions.

A Product Management Specialist will be in contact via email to discuss the specifics and technical aspects of your project.

Best regards,
Elizabeth

I want to synthesize short mRNA in vitro using DNA templates. Which genes should I use to be transcribed to a short functional mRNA? -Dela

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Dear Dela,

Thank you for your inquiry. I believe that you are referring to the elements needed in your transcription template to create a functional mRNA. These are located upstream or downstream from your open reading frame (gene). If this is what you are referring to, I have listed the elements below.

Your mRNA transcription template must include:
1. Promoter, preferably the T7 promoter
2. 5′ untranslated region with a strong Kozak sequence
3. ORF beginning with a start codon and ending with a stop codon
4. 3′ untranslated region
5. Poly(A) track top strand (this is not the same as a poly(A) signal)*
6. Unique restriction site at the end of the cassette that is suitable for linearization

*Most plasmids designed for expression in a eukaryotic cell contain a poly(A) signal rather than a poly (A) stretch. If you do not have a poly(A) track in your plasmid a poly(A) tail must be added through a poly(A) polymerase reaction.

If this is not the information you were seeking, please email info@trilinkbiotech.com and our technical team will assist you.

Best Regards,
Brea

Brea Midthune, Ph.D.
Business Development Analyst II

I want to order some primers for the detection of mtDNA damage in both rat and human. There are some mitoPrimers™ Available in the website of your company, such as A1, B1 or C1. I want to know that do these primers work for both rat and human? Thanks! – Yun-Hui

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Dear Yun Hui,

Thanks for asking about our mitoPrimers™. mitoPrimers™ were designed based on the Hypervariable 1 and 2 regions of human mtDNA to prevent any cross-species amplification between species.

I compared the A1 and CA mitoPrimers™ to the Rattus norvegicus mitochondrial genome and found that the A1 mitoPrimer™ aligns with two mismatched base pairs. C1 mitoPrimer™ did not align. I would recommend you compare the primer sequences to your specific rat genome using BLAST to determine if the mitoPrimers™ are compatible.

Please do not hesitate to contact me with any further questions.

Best regards,
Sabrina Shore, MS
Senior Research Associate

I would like to synthesize the following oligo: 5′-CAGGGGCCTACACGTTGTA-3′. The problem I understand is the 4 Gs (starting at base 3) in a row will potentially cause synthesis issue. Also this G-quadruplex will potentially form undesirable secondary structure for PCR applications in the presence of potassium. To mitigate, I learned that it’s possible to synthesize an oligo with a G analog, such as 7-Deaza-2′-deoxyguanosine to lessen the chance for the potential problems mentioned above. If this analog indeed is the best course of action, what’s the best position(s) to insert this G analog, and how many to insert? However, I also read inserting multiple 7-Deaza-2′-deoxyguanosine could be problematic because 7-deaza-dG is sensitive to the iodine-based oxidizer solution used in phosphoramidite-based DNA synthesis. So, a suggestion is to substitute 7-deaza-dG with 7-deaza-8-aza-dG, which I’m not sure you have available as an option. -Chun-Nan

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Dear Chun-Nan,

Thank you for your interest in TriLink. Poly guanosine and deoxyguanosine stretches have the propensity to form tetraplexes (complexes consisting of four different strands all bound at the poly G region). These complexes are very tight and make oligonucleotide synthesis difficult.

The use of 7-deaza-dG during PCR reduces the secondary structure but, unfortunately, the 7-deaza-dG and 7-deaza-8-aza-dG amidites have the same innate properties as guanosine during oligonucleotide synthesis. Synthetic and processing capabilities would still be problematic. For multiple incorporations of 7-deaza-dG, CSO is recommended as the oxidizing solution to combat degradation.

TriLink has devised specialized protocols for multiple and/or consecutive insertions of guanosine or 7-deaza guanosine. Due to the complexity of manufacturing, there is typically reduced yield or purity compared to a sequence with better base distribution. The number of incorporations of guanosine, 7-deaza-guanosine and/or 7-deaza-8-aza- guanosine will determine the final effect.

I would recommend trying the unmodified compound before proceeding with the modified version as the cost of the modifications will lead to an overall higher price.

Best regards,
Katelyn Murphy
Product Manager

Can you provide an expiration date on oligonucleotides? – Vivian

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Dear Vivian,

Thank you for your question. Unfortunately, we cannot provide expiration dates on specific oligonucleotides, as this data could differ with each individual sequence. As long as an oligonucleotide is stored at -20°C or lower for long term storage and multiple freeze thaw cycles are avoided, we have seen that dye labeled oligonucleotides can be stable up to 6 months and unmodified oligonucleotides stable up to 1+ years.

Best regards,
Elizabeth

I’m interested in using CleanAmp™ dNTPs with a one-step RT-PCR kit. In Figure 8 of your poster, it shows an example of a one-step RT-PCR reaction using CleanAmp™ dNTPs, but I do not see an activation step prior to RT at 42C. Can you comment on this? -Luke

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Dear Luke,

Thank you for your interest in our poster, Pushing the Limits of PCR, qPCR and RT-PCR Using CleanAmp™ dNTPs. An activation step is not required for RT-PCR as the CleanAmp™ dNTPs are activated by the combination of heat at 47°C and the acidification of Tris Buffer with increasing temperature. This releases enough dNTPs to generate a cDNA during the RT reaction. The slower activation helps decrease off-target reaction during both the RT and PCR steps.

I recommend using the conditions shown in Figure 9 of the poster which I have included below for your convenience. Of note, we recommend using a traditional 3-step thermocyling protocol, as it will provide more consistent performance than a faster 2-step protocol.
RT-PCR Conditions: Buffer (50 mM Tris-HCl; 75 mM KCl; 3 mM MgCl2), 0.4 mM CleanAmp™ dNTPs, 0.5 µM Forward Primer, 1.0 µM Reverse Primer, 100 U M-MLV (RNase H-) RT, 2.5 U Taq polymerase, 0.032-500 ng Target RNA, 10 mM DTT, 0.4 U/µL RNase Inhibitor, 25 µL reaction.
RT-PCR Cycling Conditions: 47°C (30 min); 94°C (10 min); [94°C (15 sec), 62°C (30 sec), 72°C (1 min)] 40X; 72°C (5 min)

Best regards,
Sabrina Shore, MS

Are there protocols for polymorphic microsatellite analysis using IRDye labeled nucleotides instead of primers? There must be some issue because I cannot find any. -Janine

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Dear Janine,

Thank you for your question. Labeled primers are typically used versus labeled dNTPs to allow for control over the number of fluorophores that are associated with each fragment. I am unaware of a protocol that uses labeled dNTPs.

If you decide to use labeled primers, TriLink offers a range of IRDyes for oligonucleotide labeling through OligoBuilder®. I’m sorry I could be of more help and good luck with your research.

Best regards,
Natasha Paul, PhD

I’d like to order dye-modified DNA. [TTTTT/{Texas-Red-X}/TTTTT] In terms of internal dye molecules, could you please let me know how the dye can bind between the sequences? And, I wonder if there is agap between front-bases and back-bases, because I want to hybridize this with other complementary DNA(A10). -Taeseok Oh

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Dear Taeseok,

Thank you for your question. You can order your dye-modified oligo through OligoBuilder®, our online ordering system.

Texas Red can be incorporated into your oligo by using a selective amino linker, like Thymidine-5-C2 Amino Linker or the C6 version. This modification can replace one of your bases and hybridize with your target.

Please let me know if there is anything else I can help you with.

Best regards,
Elizabeth

My NTP shipment arrived without dry ice. Should I be concerned?

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We ship our triphosphates on dry ice as a precaution. They are stable at room temperature, and even higher temperatures, for short periods of time. The chance of degradation during transit is very low. We recommend analyzing the material by AX-HPLC and then testing it in your application, provided the cost of the assay is not prohibitive. Contact us to request our analysis method.

How can I clone my aptamer library? Is there a commercially available vector with convenient NdeI and SpeI sites that can be used to clone your aptamer libraries?

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The pSNAPf plasmid from New England Biolabs (cat no. N9183) has an NdeI site (nt 484) and SpeI site (nt 249) that can be used for cloning. Alternatively, a PCR fragment amplified with Taq polymerase will have TA ends that can be cloned into a TA cloning vector such as TOPO® TA vector from Life Technologies.

I am interested in the stocked aptamer libraries. I have two questions, firstly do you have any publications resulting from the stocked library and secondly with the NdeI and SpeI sites, do you know of a commercially available E.coli vector which has these multiple cloning sites? Cheers, Chris

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Dear Chris,

Thank you for your questions about our Aptamer Libraries. NeoVentures Biotechnology analyzed a TriLink library to show the randomness of the library by enzymatic digest and by sequencing.

The pSNAPf Vector from New England Biolabs contains both a NdeI site and an SpeI site. Alternatively, a PCR fragment amplified with Taq polymerase will have TA ends that can be cloned into aTOPO® TA vector from Life Technologies.

Best regards,
Elizabeth

I would like to order a DNA oligo which contains 12bp random nucleotides (Ns) in the middle, if I wish to have all 4^12=16777216 possible oligos, what synthesis scale order I should place with you guys? Can you tell me how could assure that? -Lisa

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Dear Lisa,

Thank you for interest in our randomer service. The expected yield of our various scales can be found on the Phosphodiester Product Information. An 0.2 μmole HPLC purified, DNA synthesis typically yields 5-15 OD260 units which is 25-75 nmoles of final product for an oligo with an extinction coefficient of 200 OD260/μmole. 25 nmoles of product is equivalent to 1.5 x 1016 sequences. You would expect on average 9.0 x 108 copes of each of your ~1.7 x 107 possible sequences within the 25 nmole yield.

TriLink has done extensive research to optimize the “n” wobble (A, C, G, T) to achieve as close to a 1:1:1:1 equal ratio as possible in the final oligo product. We also offer an enzymatic digest assay which measures the base distribution of the final product. You can order your oligo through OligoBuilder® and include your yield requirement and if you are interested in an enzymatic digest in the Notes.

Best regards,
Natasha Paul, PhD

I want to order the following an antisense, a nonsense and two sonic hedgehog sequences. Can I order them through you? -Jennifer

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Dear Jennifer,

You can order a synthesis of each of the oligos you need through our OligoBuilder® system. OligoBuilder® will provide a price based on the scale and purification required. If you unsure of the scale, you can include the amount of material needed in the comments sections.

You can reference our OligoBuilder® Help Section or contact us if you’d like a further explanation.

Best regards,
Elizabeth

I have been trying to make mRNA through in vitro transcription while substituting U with pseudouridine and C with 5-methylcytidine. However, yields are much lower than when using all natural nucleotides, and 260/280 is 1.5 instead of about 2.0 I get when using natural nucleotides, and the RNA looks shorter on gel. I have been using the Ambion T7 megascript kit, and it works fine for natural nucleotides, I have been getting 100 – 150ug RNA per reaction. Do you have any suggestions for making it work well with modified nucleotides? – Samuel

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Dear Samuel,

Thank you for your question. The inclusion of modified bases in a transcription can alter several parameters. If you are including ARCA in the reaction and reducing the GTP (e.g. 4:1 ARCA:GTP) in order to give good capping, this will reduce your expected transcription yields by about 3-4 fold. In our hands, full substitution of U and C with pseudo-U and 5-methyl-C do no significantly reduce transcription yields. For an ARCA cappedmRNA  fully substituted with pseudo-U and 5-methyl-C, we typically see crude yields of 1.5-2 mg/ml of transcription. We optimize transcription reactions by varying the MgCl2 concentration. 4-6 mM over total NTPs usually works well.

With regards to the 260/280 ratio, the extinction coefficients and lambda maxes of pseudo-U and 5-methyl C differ from U and C. The lambda max for pseudo-U is 265 vs 262 and 5-Methyl-C is 279 vs 271. Thus, pseudo-U and 5-methyl-C substitution would be predicted to give you a higher 260/280 ratio than normal NTPs. If you are observing a lower 260/280 ratio, this might mean that you are actually doing a better job of removing protein in this prep. I would not be concerned by this.

With regard to mobility, we do sometimes see that some modifications change the mobility of the RNA on gels. Base modification can change the secondary structure of the mRNA and thus the mobility on non-denaturing gels. We recommend glyoxal treating the mRNA with NorthernMax®-Gly Loading Dye and then running it with NorthernMax® 10X Running Buffer (do not use TAE or TBE).

I hope this information is helpful. If you continue to have problems, you could considering order a custom mRNA.

Best regards,
Anton McCaffrey, PhD
Principal Scientist

Do you recommend making an OD320 or OD340 subtraction correction when calculating the concentration of an oligo solution? I am specifically interested in measuring the concentration of T16 oligo accurately. -Andrew

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Dear Andrew,

We typically not do recommend using a subtraction correction and recommend measuring the concentration of a T16 oligo at 260 nm, the wavelength at which thymidine absorbs. If you are trying to employ a correction for a specific buffer, the exact subtraction would depend upon the buffer you are using. Please contact us for additional information.

Best regards,
Elizabeth

What base modifications can be incorporated into my custom mRNA or long RNA?

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TriLink offers a variety of modified rNTPs suitable for in vitro transcription. We have assessed transcription efficiency based on final product formation of a 1.9 kb transcript with the following triphosphates at 100% substitution using T7 polymerase.

NTP Transcription Efficiency
5-Aminoallyl-CTP (N-1065) +++
2-Amino-ATP (N-1001) +++
5-Br-UTP (N-1054) +
5-Carboxy-CTP (N-1084) ++
5-Carboxy-UTP (N-1091) n/a
5-Carboxymethylester-UTP (N-1096) ++
7-Deaza-ATP (N-1061) ++
5-Formyl-CTP (N-1085) +++
5-Formyl-UTP (N-1090) +
5-Hydroxy-CTP (N-1089) ++
5-Hydroxy-UTP (N-1092) ++
5-Hydroxymethyl-CTP (N-1087) ++
5-Hydroxymethyl-UTP (N-1086) +++
5-Iodo-UTP (N-1012) +
5-Methoxy-CTP (N-1094) ++
5-Methoxy-UTP (N-1093) ++
N6-Methyl-Amino-ATP (N-1083) ++
N6-Methyl-ATP (N-1013) +
5-Methyl-CTP (N-1014) +++
Pseudo-UTP (N-1019) +++
Thieno-CTP (N-1097) n/a
Thieno-GTP (N-1088) ++
1-Thio-ATP (N-8005) +
2-Thio-UTP (N-1032) +++

+++ = Greater than 75% efficiency compared to unmodified NTP
++ = Between 25-75% efficiency compared to unmodified NTP
+ = Less than 25% efficiency compared to unmodified NTP
n/a = No significant product formed with 100% substitution

In all cases, transcription was carried out at 37°C.

I don’t see any expression after transfection. What should I do?

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We suggest validating the mRNA in an easily transfected cell line, such as HEK-293 cells. You may also want to include a GFP plasmid as a positive control. Note that while the GFP plasmid will provide confirmation of transfection, is does not verify delivery to the correct cellular compartment. The target compartment for the plasmid is the nucleus and the target compartment for the mRNA is the cytoplasm.

Which transfection reagent should I use?

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Several companies offer transfection reagents. Our collaborators have used TransIT®-mRNA Transfection Kit (Mirus),  Stemfect™ (Stemgent), mRNA-In™ (MTI-GlobalStem), RmesFect™ Transfection Reagent (Oz Biosciences), and Lipofectamine™ RNAi Max (Life Technologies) with success. We suggest testing a matrix of transfection reagents and varying ratios of RNA to transfection reagent. Just as with plasmids and oligonucleotides, the optimal transfection procedure will need to be determined empirically. Some cell types are intrinsically easy to transfect (e.g. HEK-293 cells ~97%). Efficient delivery to other cell types, such as some primary cells can be very challenging.

My experimental system contains RNases. How do I combat degradation?

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For many applications it is desirable to increase the nuclease stability of the RNA. The most common approach is to substitute canonical bases with 2’-fluoro modified NTPs. Bacteriophage polymerases do not efficiently incorporate 2’ modified NTPs. However, selection strategies have been utilized to evolve polymerases that can incorporate 2’ modified NTPs. Researchers commonly substitute pyrimidine bases with 2’ fluoro modified bases when making RNAs for biological applications, such as aptamers.

What precautions should I take when working with mRNA and long RNA?

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We recommend that surfaces are wiped down with RNase Zap® and disposable plasticware is used for all supplies and reagents that will contact RNA. Use RNase-free reagents and a fresh bottle of serum-free media for diluting RNA and lipids. Water can be made RNase free by treating with DEPC and autoclaving. Alternatively, you can purchase RNase-free reagents. If possible, dedicate a set of pipettes for RNA work and use barrier tips. Note that serum contains Rnases and will likely degrade your RNA very quickly.

I would like to supply my own transcription template. What elements are required?

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 mRNA Transcription Template:

  1. Bacteriophage promoter, preferably the T7 promoter
  2. 5′ untranslated region with a strong Kozak sequence
  3. ORF beginning with a start codon and ending with a stop codon
  4. 3′ untranslated region
  5. Poly(A) track top strand (this is not the same as a poly(A) signal)*
  6. Unique restriction site at the end of the cassette that is suitable for linearization

*Most plasmids designed for expression in a eukaryotic cell contain a poly(A) signal rather than a poly (A) stretch. If you do not have a poly(A) track in your plasmid we can add a poly(A) tail through a poly(A) polymerase reaction.

If your plasmid does not contain one or more of these elements, please contact us.

Long RNA Transcription Template:

  1. Bacteriophage promoter, preferably the T7 promoter
  2. Sequence that starts with G or GCG
  3. Unique restriction site at the 3′ end of the sequence to prevent runoff transcription*

*Please note that your transcript may contain a few untemplated nucleotides on the 3′ end.

If your plasmid does not contain one or more of these elements, please contact us.

What information is required for a custom mRNA or long RNA synthesis quote?

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Step 1: Identify the Source of Transcription Template:   There are two primary options for the template source. We can synthesize and clone your sequence into our specially designed plasmid or you may submit your own template (PCR product or plasmid). The template must contain the elements as described in the ‘Transcription Templates’ section.

Step 2: Provide the Sequence: If you choose to have us synthesize and clone into our specialized plasmid, we need to know the sequence you would like to express.

Step 3: Identify a Transcription Scale:  Yield estimations below are based on a 1-2 kb transcript.

Transcription Scale Uncapped RNA Expected Yield Capped RNA Expected Yield
0.5 mL 0.5 – 1.5 mg 0.25 – 0.75 mg
1.0 mL 1 – 3 mg 0.5 – 1.5 mg
2.0 mL 2 – 6 mg 1 – 3 mg
4.0 mL 4 – 12 mg 2 – 6 mg
6.0 mL 6 – 18 mg 3 – 9 mg
10 mL 10 – 30 mg 5 – 15 mg

*Please inquire for smaller scales. TriLink also has the capability to do high throughput, small-scale RNA transcript synthesis.

Step 4: Select Modifications:  Tell us if you would like modified bases in your mRNA such as a 7-methylguanosine cap, 2-thiouridine, pseudouridine and 5-methylcytidine.

Step 5: Determine Number of Constructs:  It is much more economical to order the simultaneous synthesis of several RNAs.

 

For the 3′ biotin TEG modification, is the biotin TEG directly off the 3′ end? Or is there an intervening phosphate, such as is pictured for the 5′ modification? If not, is it possible to have the 3′ end first phosphorylated and then modified with the biotin TEG? Thanks for your help! -Molly

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Dear Molly,

Thank you for your questions. The 3′ Biotin TEG modification does include a phosphate group between the TEG linker and the first nucleotide in your sequence. We look forward to helping you with your project!

Best regards,
Elizabeth

Is it known whether 7 deaza dGTP will interfere with cytosine methylation by DNA Methyltransferase, which recognise CG dinucleotides? -Aaron

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Dear Aaron,

A paper by Clark et. al. which studied the affect of different analogs on DNA methyltransferase showed that while dG and 7-deaza-dG will inhibit methyltransferase to a similar extent, they did not review the scenario where dC is upstream from 7-deaza-dG. The findings show that dG and 7-deaza-dG have similar properties and therfore, dC followed by 7-deaza-dG may be tolerated by the DNA methyltransferase. The only sure answer can be found by experimental testing.

Best regards,
Elizabeth

I recently bought carboxyl dCTP and tried with different polymerases however I was unable to amplify my construct. I tried also C, meC, hmC, fC and they worked fine with AccuPrime, but nothing to do with caC. What do you suggest? Any advice on which polymerase I can use? -Robert

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Dear Robert,

Thank you for contacting TriLink. I am not aware of any specific enzyme which is known to incorporate 5-carboxy-dCTP however; since different analogs incorporate with different efficiencies, I recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions. Alternatively, decreasing the elongation temperature, increasing elongation time in PCR cycle, increasing concentration of dGTP and 5-carboxy-dCTP or adjusting the pH may support incorporation. Please keep in mind this modification is sensitive to pH and should be kept in basic conditions. Do not hesitate to contact us if you have any further questions.

Best regards,
Elizabeth

How do I know which mitoPrimers™ to use for sequencing?

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The A1/B1 and C1/D1 PCR products can be sequenced using primers A1/A2/A4/B1/B2/B4 and C1/C2/D1/D2/D4, respectively. As a first step, use primers A1/ B1 and C1/D1 for sequencing unless there are problems in getting a continuous sequencing read. Should this problem be encountered, more focused sequencing reads can be performed using primers A2/A4/B2/B4 and C2/D2/D4.

What is the DADE linker and what are its advantages over other carboxyl linkers?

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The DADE linker is a pre-activated carboxyl linker for the 5’ terminus of an oligonucleotide. It was designed for the solid phase conjugation of amine bearing compounds directly to an oligonucleotide, although it has other applications. If it is left in its activated form when the oligonucleotide is deprotected, it will react with the prevalent nucleophile in the solution (ammonia in ammonium hydroxide forming the amide, the hydroxyl in sodium hydroxide forming carboxylic acid, etc.). Its major advantage is that it can be used for solid phase conjugation to amine bearing compounds, negating the need for costly succinimidyl esters. This allows high throughput screening, and the reuse of compound that did not conjugate in the first reaction. It also allows for the use of large excesses that will enhance conjugation efficiencies, and reduce cost. It has many other applications.

What is the difference between the TFA C6-Amino Linker and the MMT C6-Amino Linker?

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The main difference is the protecting group. The MMT linker is protected with a monomethoxytrityl group and the TFA linker is protected with a trifluoroacetate group. The MMT linker is acid labile, whereas the TFA group is base labile. MMT is the best choice when you want a pure amino labeled oligonucleotide. Since the MMT group is more nonpolar, it acts as a purification handle on reverse phase HPLC just like the DMT group. The TFA protected linker is better for crude conjugations where the oligonucleotide is used after deprotection. As a word of caution, remember to exchange the ammonia for another salt prior to conjugation since excess ammonia will severely reduce the efficiency of many conjugations.