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.
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.
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
|CleanTag™ Ligation Kit
|CleanTag™ 3′ Adapter
||CleanTag™ 3′ Adapter
|CleanTag™ 5′ Adapter
||CleanTag™ 5′ Adapter
|Enzymes, 1 & 2
||Enzymes, 1 & 2
|Buffers, 1 & 2
||Buffers, 1 & 2
|dNTP Mix (10 mM ea)
|High Fidelity PCR Master Mix (2X)
The CleanTag™ Ligation Kit was discontinued in early 2016.
We recommend a two step magnetic bead purification using Agencourt AMPure® XP – PCR Purification (A63880) by Beckman Coulter. The protocol can be found on the product insert for the CleanTag™ Small RNA Library Prep Kit (Cat# L-3206).
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.
We do provide fluorescently labeled nucleotides. Click Here for a list of our catalog dye-labeled nucleotides.
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.
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.
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.
Dear Lee Hye,
Thank you for your question. We lyophilize our oligonucleotides in a lyophilizer or speed vac. Please let us know if you have any additional questions.
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.
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.
9. Concentrate ~3X with 10K centrifugal filter device.
10. Add back 3X volume H2O.
11. Repeat steps 9-10 at least 6 times.
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.
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
Product Specialist I
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.
We highly recommend that synthetic, modified long RNAs intended for biological applications are PAGE and HPLC purified. PAGE is better at resolving long, synthetic RNAs while HPLC is critical for removing trace impurities leftover from the PAGE purification process.
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.
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.
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.
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:
For a formal quotation, please contact us through firstname.lastname@example.org
Product Specialist I
mRNA offers several advantages over traditional plasmid and viral-based approaches:
- 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.
- mRNA serves the dual purpose of expressing the desired antigen as well as acting as an adjuvant.
- 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.
- 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.
- 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.
- mRNA is easily customizable. The ease of manufacturing makes it a viable option for personalized treatments.
While we recommend sticking to 2 uL of RNA input when possible, we found that we still obtain good library prep results with up to 10 uL of RNA (without changing any other volumes).
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.
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.
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.
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.
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.
Sabrina Shore, MS
Buffer components may cause higher base pair shift in crude Bioanalyzer® traces as show below. miRNA peak should be at ~150 bp for crude samples and at ~140 bp when purified. piRNA peak should be at ~ 160 for crude samples and at ~150 bp when purified.
Diluted Crude Sample
Our CleanTag™ Small RNA Library Prep Kit (Cat# L-3206) contains adapters compatible with the Illumina® sequencing platform, however you can easily convert your tagged library into Ion Torrent™ compatible sequences during the PCR step. Contact us to discuss.
The CleanTag™ Small RNA Library Prep Kit (Cat# L-3206) has been specifically formulated to work with CleanTag™ adapters. We strongly recommend using our workflow to ensure best results.
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.
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.
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.
Sabrina Shore, MS
Scientist, Research & Development
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.
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.
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.
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.
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.
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.
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.
Yes. Some optimization may be required in your system.
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.
The RT enzyme has an optimal temperature range of 42°C – 48°C. Due to the CleanAmp™ deprotection kinetics, we recommend temperatures at the higher end of that range, optimally 47°C.
Yes. We have successfully amplified up to 4 targets in the same reaction. Higher plexing may be possible.
Yes. In addition to primers, template RNA and water, add real-time reagents according to manufacturer’s recommendations (SYBR® Green, SYTO® 9, TaqMan® probes, ROX™, etc.)
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.
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.
Sabrina Shore, MS
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.
Under certain conditions mRNA may precipitate, particularly at high concentrations. Try heating the mRNA for 15 min at 37°C to improve solubility. Long incubations at elevated temperatures should be avoided.
mRNA can have solubility issues at high concentrations. This is particularly true if the sequence is codon optimized, as with TriLink’s Cas9 mRNA. Heating the mRNA for 15 min at 37°C may improve solubility. Long incubations at elevated temperatures should be avoided.
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.
Dear Dr. Xusheng Qiu,
Thank you for your inquiry. We can prepare our triphosphates as the Li+, Na+ or TEA salt form. I believe our dDTP is currently in the Li+ salt form. I will have our technical support team contact you regarding preparing the Na+ salt form.
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.
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.
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 email@example.com and our technical team will assist you.
Brea Midthune, Ph.D.
Business Development Analyst II
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.
Sabrina Shore, MS
Senior Research Associate
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.
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.
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)
Sabrina Shore, MS
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.
Natasha Paul, PhD
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.
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.
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.
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.
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.
Natasha Paul, PhD
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.
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.
Anton McCaffrey, PhD
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.
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.
+++ = 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.
You can confirm translation by western blot or enzyme-linked immunosorbent assay (ELISA).
We have assessed activity of our stocked EGFP in a variety of cell types, including HEK-293, RAW 264.7 and BJ Fibroblast cells. Additionally, we have expressed other stocked mRNA in HEK-293, CHO, BJ Fibroblasts, CEM and primary human CD34+ cells.
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.
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.
Our standard purification consists of two silica column purification steps. If you are interested in alternative purification methods, please contact us.
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.
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.
Long-term storage should be between -40°C and -80°C with limited freeze-thaw cycles.
Transcript length and integrity are confirmed via agarose gel analysis after glyoxal treatment. Quantity and purity are determined through ultraviolet spectroscopy.
Each template is unique and it is very difficult to predict the outcome of a transcription. Elements such as strong hairpins and repetitive sequences can cause transcriptional stops. If this is a concern, we will perform a small scale transcription and work with you to optimize the transcription reaction for your particular template if needed.
For stocked items, we elute in 10 mM Tris-HCl, pH 7.5. For custom syntheses, we elute in RNase-free water, however we are able to accommodate most special requests.
Our standard purification consists of two silica column steps. We also offer HPLC and PAGE purification.
mRNA Transcription Template:
- Bacteriophage promoter, preferably the T7 promoter
- 5′ untranslated region with a strong Kozak sequence
- ORF beginning with a start codon and ending with a stop codon
- 3′ untranslated region
- Poly(A) track top strand (this is not the same as a poly(A) signal)*
- 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:
- Bacteriophage promoter, preferably the T7 promoter
- Sequence that starts with G or GCG
- 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.
Order stocked mRNA directly through our website.
For a custom synthesis, request a quote online.
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.
||Uncapped RNA Expected Yield
||Capped RNA Expected Yield
||0.5 – 1.5 mg
||0.25 – 0.75 mg
||1 – 3 mg
||0.5 – 1.5 mg
||2 – 6 mg
||1 – 3 mg
||4 – 12 mg
||2 – 6 mg
||6 – 18 mg
||3 – 9 mg
||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.
TriLink’s stocked mRNA products have been optimized for expression in mammalian cells and organisms. Activity in non-mammalian cells (i.e. flies, fish and worms) has not been evaluated. If you achieve expression with one of our stocked mRNA products in cells from other species, please share your results with us.
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!
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.
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.
Use the A1/B1 and C1/D1 primer sets to PCR amplify human mitochondrial DNA hypervariable region 1 (HV1) and hypervariable region 2 (HV2), respectively.
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.
mitoPrimers™ undergo a proprietary purification process to ensure high-quality sequencing data. Furthermore, mitoPrimers™ have been quality control tested with NIST-certified DNA in a PCR/Sanger sequencing assay, thereby allowing less in-house validation by the end-user.
CleanAmp™ dUTP is a dUTP analog which contains the CleanAmp™ thermolabile protecting group for improved PCR specificity.
Optimal PCR results are achieved when CleanAmp™ dUTP is in a three-fold excess over dATP, dCTP and dGTP. Use CleanAmp™ dUTP with standard dNTPs for well-behaved targets and with CleanAmp™ dNTPs for problematic targets.
CleanAmp™ dUTP can be used with uracil-N-glycosylase (UNG) in PCR-based carryover decontamination schemes. Please refer to the enzyme manufacturer for the recommended conditions for the UNG step of the reaction.
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.
Yes, click here to view the reagents still sold through TriLink. All of our other DNA synthesis reagents are now sold through Glen Research.
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.
CleanAmp™ 7-deaza-dGTP and CleanAmp™ 7-deaza-dGTP Mix function well on their own for most sequences with <80% GC-content. However, should you have a problematic target or one that is >80% GC-content, addition of any one of these additives may increase specificity. We have found 0.5M Betaine or 10% Glycerol give the best results.
Preliminary results show CleanAmp™ 7-deaza-dGTP Mix functions with a variety of enzymes. However, we recommend Invitrogen™ Taq DNA polymerase for best results. For questions regarding your specific enzyme, please contact us.
CleanAmp™ 7-deaza-dGTP Mix contains the CleanAmp™ modified nucleoside triphosphates of dA, dC, (dG:7-Deaza-dG) and dT at 10 mM. The dG mix is a 1:3 ratio of CleanAmp™ dGTP:CleanAmp™ 7-deaza-dGTP.
For targets with <70% GC-content, addition of theCleanAmp™ 7-deaza-dGTP with standard dNTPs should amplify the target well. For targets with >70% GC-content, we recommend theCleanAmp™ 7-deaza-dGTP Mix for a robust amplification.