不同标记荧光基团的稳定性、半衰期介绍说明

Please refer to STYLE GUIDE.doc for detailed guidelinesColor code legene:Red = Proprietary; Pink = Discontinuation; Green = Anecdotal; Blue = Anything else customers will not see Custom Primers – All ModificationsTABLE OF CONTENTSPRODUCT DESCRIPTIONSHIPPING CONDITIONSSTORAGE CONDITIONSSTABILITYQC SPECIFICATIONSPROTOCOL & APPLICATION NOTESModification/scale/purificationManufacturing DetailsFluoresceinRhodamineHEX/TET/FAMPhosphateBiotinAmineGatewayAlexaOther dyesAlkaline PhosphataseHorse Radish PeroxidasePhosphorothioate“Fully-Phosphorothioated" Custom PrimersAldehydeAcridineThiolDelivery ScheduleOligoPerfect Designed Primers3' Modifications of OligosReconstitution ProtocolA260/A280 ratio of the oligoOligo VisualizationTroubleshooting“Custom Custom” modificationsList of current technology limitationsCOMPETITOR INFORMATIONALTERNATE PRODUCTS & COMPATIBILITYPRODUCT DOCUMENTATIONREFERENCESPRODUCT NAME & CATALOG NUMBERCOMPONENTSDISCONTINUATION NOTICEASSOCIATED PRODUCTSPRODUCT QUALITY ISSUESReturning PrimersLICENSINGINTERNAL CONTACTSPRODUCT DESCRIPTION(back to Table of Content)Custom DNA Primers are synthetic oligonucleotides with specified sequence made to order for use in a variety of applications from PCR and sequencing to probes for gene detection. Custom DNA Primers are available as standard deoxynucleotides, modified bases, 5´ modified nucleotides (including fluorescent dyes, enzyme conjugates), and S-oligos for antisense studies. Five scales and four purity levels are available.A unique and proprietary technology and an optimized production process is used to deliver quality Custom Primers, quickly and efficiently. The process includes easy ordering, linkage to integrated workstations for guaranteed accuracy, high-throughput synthesis, monitoring by a series of stringent quality control steps, useful product packaging, and overnight shipping for rapid, reliable delivery. Custom oligonucleotides are synthesized on automated, proprietary DNA synthesizers using standard cyanoethyl phosphoramidite chemistry. Consistent oligonucleotide quality is ensured by in-process trityl monitoring and quality control analysis. A comprehensive Certificate of Analysis accompanies each order, indicating quantity, molecular weight, extinction coefficient, sequence, and melting temperature.DNA oligos can be ordered in tubes or plates:•Tubes:o Standard tube with cap (Scientific Specialties Inc. (SSI) 2680-00 (tube), 2001-00 (cap)). Volume is 2000µl •Plates:o96-wells:PCR shallow 96-well plate (Abgene AB-0800): 200µl max volume, 180µl working volume; plate code 96N – 180. Shape of wells: VStandard shallow 96-well plate (Corning Costar 3359): 360µl max volume, 250µl working volume;plate code 96B – 250. Shape of wells: U/round. OLD SHALLOW PLATEStandard medium 96-well plate (Abgene AB-0765): 800µl max volume, 650µl working volume; plate code 96L – 650. Shape of well: conical.Cluster tube screwtop 96-well plate (Matrix ScrewTop TrackMates 3742, ): 1000µl max volume, 750µl working volume; plate code 96D – 750. Shape of well: Unknown.SPECIAL IStandard deep 96-well plate (Thompson Instruments, Oceanside distributor, part number 951651B): 1200µl max volume, 800µl working volume (equivalent to Abgene AB-0564); plate code 96C – 800.Shape of well: U/round. OLD DEEP WELL PLATECluster tube capmat 96-well plate (Matrix TrakMates 3791, ): 1400µl max volume, 850µl working volume; plate code 96J – 850. Shape of well: V. SPECIAL II o384 wells:Standard 384-well plate – Thompson Instruments (Oceanside distributor) part number 931504B, 120µl max volume, 75µl working volume (equivalent to Abgene AB-0781); plate code 384E – 75. Shape ofwell: pyramidal.Microarray 384-well plate (Genetix X7020): 65µl max volume, 45µl working volume; plate code 384F – 45. Shape of well: flat.o Seals and caps:Heat Seal – Thompson Instruments (Oceanside distributor) part number 899406 (equivalent to Abgene AB-0745)Adhesive Seal – Thompson Instruments (Oceanside distributor) part number 899405-1Caps – Matrix ScrewTop caps 4472, for use with cluster tube screwtop 96-well plate, 96 caps required for each plateCapmat – Matrix SepraSeal Cap Mats 4463, for use with cluster tube capmat 96-well plate, one mat required for each plateSHIPPING CONDITIONS(back to Table of Content)Please refer to Delivery Schedule.PrimaryRoom Temperature lyophilized for tubes, different options for plates (lyophilized, ambient in solution, frozen in dry ice. SecondarySpecial Handling by request: DNA oligos ordered in plates can be requested to a variety of normalization, pooling and aliquoting options free of charge. For TUBE oligos, the standard format is dried down and ship at room temperature. However, oligos ordered in tubes can be requested to be sent with a few pre-selected special handling options. These options are available in the check out section of the online cart. Two of these options can be checked for an extra $1 charge per primer. The options are re-suspension of the oligos in water to a certain concentration (10, 20, 50, 100 or 200 µM), electronic COA, second set of labels, 100% QC (for DNA oligos we only do Mass Spec or CE for 25% of the oligos, this options will make the oligos go through CE/Mass Spec for sure), ship complete order and not partial orders and/or send on dry ice. For other options, a special handling request can be requested through the Invitrogen Account Manager. Re-suspended primers also ship at Room Temperature (they are stable this way), unless instructed otherwise in the “special handling”.Note: The primers from Frederick are shipped held in a cardboard holder called a “Fluted Partition”. This is made by a company called Fluted Partition (203-368-2548). The tube primers from Illumina are sent in a tube holder that holds 6 tubes.HRP Oligo shipping: The storage conditions are 4oC in HRP buffer. We ship on ice packs. The box should be marked with a sticker to refrigerate upon arrival.STORAGE CONDITIONS(back to Table of Content)Recommended storage for lyophilized primers and reconstituted primers is -20°C.AP Conjugate is shipped in AP storage buffer and should be stored at 4°C.HRP Conjugate is shipped in HRP liquid storage buffer on ice packs and should be stored at 4°C.HEX, TET and FAM are very sensitive to light. Store in the dark or use amber tubes. Storage in a black box is recommended. HEX and TET labels are both stable under 30 cycles of standard PCR conditions; however under harsher conditions (high pH, high temp) TET is more stable than HEX.Dye half life (approximately) when exposed to lightHEX 4.5 hoursTET 2.25 hoursFAM 1.125 hoursFor making adapters with 5’modified oligos:We have limited data to show that in high salt solution at 55°C HRP is stable for 1 hour.We have no stability data of HRP at 65°C. It has been stated that HRP is less stable than APSTABILITY(back to Table of Content)Lyophilized primers are stable for one year, and will be stable for several months at 4°C. Reconstituted primers at -20°C are stable for at least six months, and they will be stable for a few weeks at 4°C.AP Conjugate - shipped in AP storage buffer, store at 4°C, stable for at least 12 months.HRP Conjugate - shipped in HRP storage buffer, store at 4°C, stable for at least 12 months.QC SPECIFICATIONS(back to Table of Content)Most of the modified oligos arel be made at Frederick (see General Custom Primer note for details).For 25, 50 and 200 nM desalted and cartridge-purified DNA oligos, there is 100% OD 260 analysis. Random samples of 25% of the oligos produced are tested by either Capillary Electrophoresis (all oligos at Illumina and any oligo longer than 45-mers at Frederick) or Mass Spectrometry (Illumina does not do Mass Spec anymore but Frederick still uses it for oligos smaller than 45-mers). DNA oligos that are desalted and ordered at 25 and 50 nM scales also have 100% real-time digital trityl monitoring during analysis. Customers can request 100% QC by Mass Spec or CE during ordering paying $1 per oligo.Desalted DNA oligos ordered at 1 and 10 uM, DNA oligos at any scale that are purified by HPLC and PAGE, the majority of the DNA oligos with 3 and 5’ modifications and the RNA oligos have also 100% OD260 analysis and Mass Spectrometry or Capillary Electrophoresis.For a PAGE oligo, regardless of length, the main peak on the trace needs to be at least 85% of the product to pass and the N- peaks for mutations must be less than 10% (Jaime 11.30.06, from Sheryl Moles).Effect of modification on ODOligos are quantified by measuring the absorbance at 260 nm. Some fluorophores do absorb at 260 nm as well. For example, fluorescein and TAMRA have an extinction coefficients of 20.96 and 31.98 OD/µmole at 260 nm. For reference, dA, dC, dG and dT are 15.3, 7.4, 11.8, 9.3 OD/ µmole respectively. So, TAMRA is equivalent to about 3 nucleotides. For the dyes that have a published values at 260 nm, the value is incorporated into the extinction coefficient calculation on the C of A. These modifications are: FAM, Fluorescein, HEX, TAMRA, and TET. Others may absorb at 260 nm, but the data is not published and therefore is not included in the calculation.OD Specifications for AP and HRP Conjugates- Not applicable because pass/ fail based on moles of conjugate primers. The protein and oligo make up the OD value and may not reflect the true OD value of the oligo.AP and HRP ConjugatesBases Scale Desalted Cartridge*HPLC PAGE<20 Bases25 nmol NA NA NA NA50 nmol NA NA NA NA200 nmol NA NA NA NA1 µmol NA NA1-5 nmole (0.5-13OD)1-5 nmole (0.5-13 OD)10 µmol NA NA Inquire Inquire 20 Bases25 nmol NA NA NA NAor greater50 nmol NA NA NA NA200 nmol NA NA NA NA1 µmol NA NA1-5 nmole (0.5-13OD)1-5 nmole (0.5-13 OD)10 µmol NA NA Inquire Inquire*Per Jeremiah Mitchell (03/18/2009) we can't offer any mod on a cartridge purified oligo. The modification interferes in some way with the purification mechanism.QC on Taqman probes:Taqman and other Fluorescent probes will be QC’d by Mass Spectrometry (Mass Spec, MS). “They will be run on mass spec. We try to hit the guaranteed amount but we do not make a guarantee.” – Jeremiah Mitchell (04/02/2009)PROTOCOL AND APPLICATION NOTES(back to Table of Content)Modification/scale/purificationManufacturing DetailsFluoresceinRhodamineHEX/TET/FAMPhosphateBiotinAmineGatewayAlkaline PhosphataseHorse Radish PeroxidasePhosphorothioate“Fully-Phosphorothioated" Custom PrimersAldehydeAcridineThiolDelivery ScheduleOligoPerfect Designed Primers“Fully-Phosphorothioated" Custom Primers3' Modifications of OligosReconstitution ProtocolA260/A280 ratio of the oligoOligo VisualizationTroubleshooting“Custom Custom” modificationsModification, Scale of Synthesis/Purification and Availability Grid (back to Table of Content)(back to Protocol and Application Notes)Mod SynthScale PurificationDesaltedPurificationCartridgePurificationHPLCPurificationGel Purified5' Biotin25 N Yes (10-50 bases)NO NO NO5' Phosphate50 N Yes (10-50 bases)NO NO NO5' Primary200 N Yes (10-50 bases)NO Yes (7 – 60 bases)Yes (7-100 bases) Amine 1 U Yes (10-50 bases)NO Yes (7 – 60 bases)Yes (7-100 bases)25 U Yes (10-50 bases)NO Yes (7 – 60 bases)Yes (7-100 bases)25 N NO NO NO NO50 N Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases) 5' Rhodamine200 N Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)1 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)25 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)25 N Yes (10-50 bases)NO NO NO5' HEX/ TET/FAM50 N Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases) 5' Fluorescein200 N NO NO Yes (7 – 60 bases)Yes (7-100 bases) 5' GATEWAY 1 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)25 U Yes (5-100 bases)Yes (7 – 60 bases)Yes (7 – 60 bases)Yes (7-100 bases)25 N NO NO NO NOAP Conjugate50 N NO NO NO NOHRP Conjugate**200 N NO NO NO NO1 U NO NO Yes (7 – 60 bases)Yes (7-100 bases)25 U NO NO Yes (7 – 60 bases)Yes (7-100 bases)25 N NO NO NO NO50 N Yes (5-100 bases)Yes 7 – 60 bases)YES (7 – 60 bases)YES (7-100 bases) Phosphorothioates200 N Yes (5-100 bases)Yes (7 – 60 bases)YES (7 – 60 bases)YES (7-100 bases)1 U Yes (5-100 bases)Yes (7 – 60 bases)YES (7 – 60 bases)YES (7-100 bases)25 U Yes NO YES (7 – 60 bases)YES (7-100 bases)Reason for lower limit of 10 mer on the 25 nmol scale: "We have had a high failure rate on synthesis of short oligos at the 25 nmoles scale. We have determined that the problem cannot be corrected. Therefore, we have decided to make the minimum length at the 25 nmole scale to be a 10 mer.The amount of modified oligo specified in the CoA includes the modification. For example, for the biotin modification, the amount of oligo in ‘ug’ and ‘uM’ specified in the CoA includes the oligo, the C6 linker and the biotin.**HRP max oligo length of 25 bases, requires 1 umole scale.Manufacturing Details(back to Table of Content)(back to Protocol and Application Notes)These modifications are added as phosphoramidites on the machine during synthesis.FluoresceinRhodamineHEX/TET/FAMPhosphateBiotinAmineGatewayAlkaline PhosphataseHorse Radish PeroxidasePhosphorothioateAldehydeAcridineThiol5’ FLO Modification (Formerly referred to as Fluorescein)(back to Table of Content)(back to Protocol and Application Notes)(back to Manufacturing Details)5(6)-FAM (5(6)-carboxyfluorescein) is added as a phosphoramidite by B-cyanoethyl chemistry and therefore added on the sugar at the 5' end of the primer NOT on the last base as the last synthesis cycle. This is a mixture of two isomers of FAM (therefore on an HPLC trace, there will be two peaks).Absorption Max (nm)Emission Max (nm)Extinction Coeff.(OD/mole) at maxExtinction Coeff. (OD/mole) at260 nm49452076,00031,500 Fluorescent color is green.Note: 5’ FLO is a mixture of the 5- and 6-isomers of FAM. They are not stereoisomers. The conjugation bond is either on the 5 and 6 position on the phenyl ring. Half the molecules have the bond at the 5 position and half have it at the 6 position**. The two different oligos can be detected when analyzed by HPLC or CE. The extinction coefficients at lambda max and at 260 nm are estimates based on known values of the two isomers. This modification is not available desalted or cartridge purified per Sheryl Moles 3/29/07 (V.Gurtu).The phosphoramidite used for this does not have a trityl group, but the FAM itself is hydrophobic enough to allow HPLC purification. A modified HPLC protocol is used for the 5' phosphate oligos to achieve the separation. Typically, 5' fluorescein oligos will not be as pure as those that can be purified by using the trityl on approach, but close in purity.FAM and Fluorescein are different chemicals. FAM is 5-carboxyfluorescein or 6-carboxyfluorescein (single isomers) or 5(6)-carboxyfluorescein (mixed isomers) – FAM has two carboxyl moieties on the phenyl ring, while Fluorescein has only one carboxyl on the phenyl ring; all reactive groups will be conjugated via the 5- or 6- carboxyl group on FAM but directly to the phenyl ring on Fluorescein. Although these two dyes have very similar spectral properties, the names “FAM” and “Fluorescein” are not interchangeable. FAM, not Fluorescein, is used on ABI sequencers. The two isomer forms of FAM differ in electrophoretic mobility and both FAM isomers differ significantly from the electrophoretic mobility of Fluorescein.Specs for FLO/FAM mods:“We will use 5'FLO amidite (6-FAM)so there is no free dye.If the oligo is desalted it has to have at least 45% full length product (note that these specs are not public knowledge) and contain no N- peaks that are greater than 15%.” – Jeremiah Mitchell (05/19/2009)*[Note: Highly concentrated solutions and the solid forms of fluorescein and FAM appear by eye as yellow or orange colored solutions or solid, this may also be true of lyophilized oligos labeled with 5’FLO].** “Half the molecules have the bond at the 5 position and half have it at the 6 position”. Our mixed isomer FAM products, 5(6)-FAM, can have a lot-to-lot variablility in 5:6 isomer ration from 70:30 to 50:50; is the 5(6)-FAM phosphoramidite SPECIFICALLY provided always a 50:50 ratio of isomers, or is there lot-to-lot variability as well? It is possible, so you can not always tell customers it is 50:50. (O. Cholewa, 8/2008).This modification can be added to phosphorothioated oligos.Rhodamine(back to Table of Content)(back to Protocol and Application Notes)(back to Manufacturing Details)Can suggest as the alternative to 5’ROX modified oligos.All the rhodamine dyes (rhodamine, rhodamine green dye and rhodamine green-X) are HPLC purified as part of the dye price. The web site forces you to order it as desalted for now (8.4.06) but it will be changed in the future.Rhodamine when resuspended in water or TE solution will appear pink-red-purplish.Rhodamine = RHD on email order form.MW(NH4 salt)MW (no salt)EmissionAbs. (nm)Extinct. Coeff. (at lambda max)**Max (nm)743.8726.859657292,000Note that this is for measurements of the labeling dye in methanol. The values in water/TE and attached to an oligo will be different.** We do not have info on what A260 of rhodamine would be.The modification provided is x-rhodamine isothiocyanate (X-RITC) which is a mix of rhodamine-5 and 6-isothiocyanate (5(6))-XRITC). The addition of the rhodamine modification is done on all scales as a post-synthesis modification. To see the structure, click on the link in this paragraph.HEX, TET or 6-FAM(back to Table of Content)(back to Protocol and Application Notes)(back to Manufacturing Details)HEX is 4,7,2`4`5`7` hexachlorofluoresceinTET is 2`7`4,7, tetrachlorofluoresceinFAM is 6-carboxyfluorescein, while fluorescein is the 5 and 6 isomers mixed together.FAM, HEX and TET are added as a phosphoramidite as the last synthesis cycle using B-cyanoethyl chemistry and therefore added on the sugar at the 5'end of the primer NOT on the last base. They are covalently attached to the 5' end of the last sugar via a phosphodiester bond.Point at which modification occurs:In synthesis we can add HEX, TET, FAM or flourescein. All other dyes are added post-synthesis. In synthesis mods do not require purification. (Jeremiah Mitchell, Melissa Almeida; 04/30/2009).Trityl-OFF cartridge purification on confirmation sheet refers to the fact that there is no DMT on the phosphoramidites with the dye.For Eurogentec orders take the MW from the CofA and add the below MW numbers. PrimerTrak includes the MW of the Mod in the MW from the CofA.MW Emission Max(nm)Excitation (Abs)Max (nm)Extinction (at max)Extinction (at 260)FAM554.551749474,85020,960TET692.253852285,55316,255HEX761.155353595,69831,580Molar extinction coefficients above are measured at excitation maximum nm.Individual experiments may vary fluorescence intensity due to minor variations in pH or composition can affect the above numbers. For FAM, take the reading at pH 9 since at this pH the absorbance is highest. At pH 7, this value is reduced by 16% for FAM. And when the fluorophore is attached to DNA, the value is also reduced.We do not know the effect of pH on the absorbance of TET & HEX.At the maximum wavelength, there is a huge change between pH 8 and pH 6.5. For the absorbance at 260, the change between pH8 and pH 6.5 is relatively minor.I tested the water here and it is about 6.5.Also please see /handbook/figs/fig23-2.htmlfor more information on effect of pH.The color of the dye depends on the "Filter-wheel set" of the ABI instruments:Dyes Filter A Filter BHEX Green (560nm)Yellow (560nm)6-FAM Blue (531nm)Blue (531nm)TET-Green (545)Color in solution: HEX is pinkish, FAM the yellow, and TET is orange, but if the oligos are not subjected to light they may appear colorless. 10nm often will have no color due to the small amount of oligo present.The phosphoramidites with these dyes do not have a DMT (trityl) group. They therefore cannot be monitored during the synthesis with trityl analysis. They still must be deprotected, as there are protective groups on the bases in the oligonucleotide. A & C have a benzoyl protective group. G has an isobutyl protective group. T does not have a protective group.Trityl-OFF cartridge purification is required since these do not have a DMT. This purification is performed as a batch HPLC method on a polystyrene reverse phase support.They cannot be ordered as phosphorothioates.Phosphate(back to Table of Content)(back to Protocol and Application Notes)(back to Manufacturing Details)5’- PhosphateThe cartridge purification option for the 5' phosphate oligos is not offered, because it is not technically possible (cartridge purification relies on the DMT group being present which is not true here). DMT has molecular weight of 79.98.The 5' phosphorylation is added by B-cyanoethyl chemistry and therefore added on the sugar at the 5'end of the primer NOT on the last base, as the last cycle with a phosphoramidite. It therefore has a trityl group, which is released and monitored to assess the coupling efficiency. (During the coupling, the phosphate has a protecting group, which is removed prior to cleavage of the oligo from the solid support). Since 5' phosphorylation comprises the final step after all the cycles are complete, only the complete, or full length oligos (which are uncapped) can be phosphorylated on the synthesizer.Gel purification of phosphorylated primers will not purify full length primers from n-1 primers because the phosphate adds an additional negative charge that affects electrophoresis.The 5’ phosphate oligos are purified by HPLC by size. A modified HPLC protocol is used for the 5' phosphate oligos to achieve the separation. Typically, 5' phosphate oligos will not be as pure as those that can be purified by using the trityl on approach. The cartridge purification option cannot be used for the 5' phosphate oligos, as this relies on the DMT group being present.In fact, when the yield of HPLC phosphorylated primers is low it probably was because there were a lot of failure sequences so not as many fractions could be combined. There would be a lot of multiple overlapping peaks in preps with more "n-x" failures. During HPLC purification, 5 Phosphate oligos are not DMT-selected.It’s been noticed that a couple of phosphate, HPLC oligos after speedvac are a bit "dirty", or slightly yellow. This is sometimes a product of the synthesized product. It is usually cleaned up in the ethanol precipitation step as part of QC.3’-PhosphateThe 3’ phosphate is added attached to the solid support so the sequence is actually coupled to it in the first round of base addition. It will block extension by polymerases. Stability: assuming it's DNA, 3'-PO4 is more stable than 3'-BIO. 3'-BIO will come off under basic condition while 3'-PO4 will not.Biotin(back to Table of Content)(back to Protocol and Application Notes)(back to Manufacturing Details)The structure as shown is called Biotin Phosphoramidite. Biotin is on the far right ending with the oxygen group next to the amine group in the chain. MW of biotin is 405. Biotin oligos are quantified as normal by OD260 (biotin does not absorb at 260). Comparing 3’ Mod Stability: 3'-NH2 is slightly more stable than 3'-PO4. It is 3'-NH2>3'-PO4>>3'-BIOThe biotin that we use for our modifications is from Glen Research, 10-5950-95 (Jeremiah Mitchell, 04/27/2009).5’- BiotinPROPRIETARY INFORMATION: It is purchased from Glen Research and Catalog number 10-5950-02./ Please see the biotin structure below.Biotin dT The Biotin dT has the Biotin is attached to a dT base, so they would only use it if the 5' base can be T. I really know no reason why one would choose it over the regular biotin.General info on Biotinylated primers:5' biotinylated primers may work for chemiluminescent detection, but TdT tailing would be better.The biotin is added during synthesis, before the purification step. A biotinylated oligo (or a PCR product made with biotinylated oligos) will be retained for a longer time on a reverse phase HPLC column than an unbiotinylated oligo of the same size. This is due to the hydrophobicity of the biotin moiety.The biotin modification should be fairly resistant to heat and pH (as it has been boiled to 100°C and exposed to pH 2-10). More extreme conditions may be tolerated, but have not been tested. Presence of reducing agents may not be a problem, but exact limits are unknown.The chemical structure of 3'-Biotin predicts that 3'-Biotin would come off/degrade at two conditions: 1 over the time, 2 the oligo was exposed to basic conditions such as dissolved in basic buffer. The reason is that 3'-Biotin has an adjacent hydroxy group which makes it a RNA-like structure.5'-Biotin though does not have this adjacent hydroxy group, so it is pretty stable, more stable than 3'-Biotin.The standard biotin that we use has a C6 linker. We have used the TEG biotin linker several times in the past because of special requests from customers but it is not our “standard” biotin modification (Jaime 5/10/06 from Joseph Hayes).Recover 5’ biotin labeled oligo from C-18 column: An oligo with biotin is more hydrophobic than an unlabeled oligo. Therefore, the biotin oligo would stick better to a C-18 column. I haven’t used a C-18 column for years since the polystyrene columns are more durable for production. To put it in perspective, on the polystyrene I elute the unlabeled oligos in 6 - 12 percentACN/TEAA. For the biotin oligos I use 10 - 15 %. The customer may be able to use this to extrapolate the percentages. Since C-18 is more hydrophobic than polystyrene, their percentages should be a little higher.Frederick QC’s the oligos by Mass Spec or CE and they check the peaks to see if the coupling reaction of the biotin to the molecule went as expected. For a DSL oligo, we will discard the oligo if there is a peak indicating more than 15% of the oligo not coupled to biotin. For HPLC or PAGE purified oligos, we will ask for less than 10% of uncoupled oligo during QC (Jaime from Sheryl Moles, 12.14.06).Biotinylated DNA will successfully transform bacteria, e.g. DH5 alpha.3’- Biotin3’ Biotin is added via biotin solid support.Primers carrying a 3’-biotin modification can not be longer than 99 nucleotides. Since the 3’-biotin is provided as a CPG modification (linked onto the solid support) the system interprets the 3’-biotin as the last nucleotide of the sequence. (Sheryl Moles 02/08/07)Comparing 3’ Mod Stability: 3'-NH2 is slightly more stable than 3'-PO4. It is 3'-NH2>3'-PO4>>3'-BIOGlen Research Catalog Number: 20-2955-xx /Primary Amine(back to Table of Content)(back to Protocol and Application Notes)(back to Manufacturing Details)Primary amine –The amino group is linked to the 5' end via a 6 carbon spacer arm. i.e. Off of the 5’ hydroxyl there is a phosphate. Attached to this via a phosphodiester bond is a C6 linker (CH2-CH2-CH2-CH2-CH2-CH2) followed by a primary amine NH2. It is added as a phosphoramidite at the last synthesis cycle by B -cyanoethyl chemistry and therefore added on the sugar at the 5'end of the primer NOT on the last base.For other scales, we can also do 3’ amine modification as a custom “custom” through PMO request (Jeremiah Mitchell). 3’ Amine mod has a C7 linker. We can now do 3’ amine modification as a catalog item for 50 nmol and 200 nmol scale. Customers will be able to order online, or using the email/fax form in North America. The website will be updated with the proper drop-downs for the mod, and the forms will be updated with the code. Please note that the 3’ amine mod is only compatible with certain 5’。