how to check if oligos are annealed

If unsure, measure the pH of your water, anything that is at least pH 6 I would use without any worries as is and do for years for DNA and RNA, even stuff with many freeze-thaw cycles. Sorry, I didnt read all your thread. Contact our Customer Service Team by Protocol for assembling annealed DNA oligonucleotides SI Abnormal Reports. OligoEvaluator for Tm Calculation & Primer Analysis <> The dss oligo will be brighter under the UV. So ideally you have filtered them first for proper length through PAGE. Milder later-adult onset forms of the disease exist presenting with neurological problems such as ataxia, dystonia, spinocerebellar degeneration, and behavior changes. Just enter you sequence in the calculator 2013 Sep;59[9]:1357-1368). For convenience, keep Annealing Buffer volume below 500 l for each oligo. I repeated the annealing and it improved a little bit but because the annealing will produce sticky ends that should in theory fit perfectly the sticky ends on the other fragments, I will just use a high amount of this linker and hope that whatever was able to anneal proper will be ligated while all the rest should be excluded from ligation. solved in TE buffer, the EDTA will basically chelate the magnesium ions and interfere with the proper folding/annealing ! I'm regularly doing long pre-synthesized inserts of several hundred bp if I have to create a de novo sequence and what Kevin mentioned is working well. WebUpdated. Prenatal onset (type III) manifests as prenatal loss or early death from progressive neurodegeneration. Available at: Disorders of glycoprotein degradation: alpha-mannosidosis, beta-mannosidosis, fucosidosis, and sialidosis. In:Valle DL, Antonarakis S, Ballabio A, Beaudet AL, Mitchell GA,eds. H}P`c6sN[O=j=rrKHby!%! Do not overlay the samples with oil. It is difficult to prevent non-somplementary binding of primers to give just 80bp dsDNA using thermocycler. The hybridized oligos or DNA duplex can be run on a non-denaturing gel with appropriate molecular weight markers. They are characterized by the abnormal accumulation of incompletely degraded oligosaccharides in cells and tissues and the corresponding increase of related free oligosaccharides in the urine. Almost all known nucleases have a zinc ion in their catalytic center. Hiiiii everyone! % DMSO is commonly used to PCR amplify GC rich sequences, and I wonder if it would be of any help here? You can do that yourself or you order them with PAGE filtering, again Thermo is doing that for example. 0000013889 00000 n generation sequencing, Genes & If you are getting smear.. then try to first heat the buffer til 70C and then add the oligos. Adult onset presents mainly with dystonia. The recommended screening test for the initial workup of a suspected lysosomal storage disorder, particularly when clinical features are nonspecific, is LSDS / Lysosomal Storage Disorders Screen, Random, Urine. The Online Metabolic and Molecular Bases of Inherited Disease. 2. pVuS.W`ym=S1I*,ky&^Fgv!5~Pd%18PU1fJHIV%L$%% 25 0 obj <> endobj xb```e``~"620 Pcd0 mD. trailer 1 ul of each oligo (100 M stock) to a final concentration of 0.2 M (0.2 pmol/l) using 1X NEBuffer r2.1*. This is a screening test; not all oligosaccharidoses are detected. In strip tubes or 96-well plate that fits a PCR cycler, add, per tube (or well): 1.5 L of forward oligo (40 M) 1.5 L of reverse Please sign back in to continue your session. The oligosaccharidoses and other storage disorders detected by this assay include alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, fucosidosis, Schindler disease, GM1 gangliosidosis, Sandhoff disease, sialidosis, galactosialidosis, mucolipidoses types II and III, mucopolysaccharidosis IVA (Morquio A), mucopolysaccharidosis IVB (Morquio B), and Pompe disease (see table). If so, try our xGen NGS Solutions Builder Tool today. 0000001615 00000 n 2/ To check the purity of long oligos and purify them it is better to do it through a denaturing Urea gel or a mini sequencing gel. Earlier for congenital, infantile, and juvenile forms (type II). 0000002329 00000 n Copyright 2023 FAQS.TIPS. I am willing to share my protocol with you if you are interested. Clinical diagnosis can be difficult due to the similarity of clinical features across disorders and their variable severity. It allows seamless cloning and is fast, very efficient and cheap. Although not always, some times in science the simplest method is the best method. Oligonucleotide phosphorylation and annealing - JBEI 4 0 obj 0000054822 00000 n Through this new partnership we are pleased to offer you comprehensive next generation sequencing solutions. 0000014645 00000 n WebUse 3 annealing reactions: 1) oligo sense only 2) oligo antisense only 3) oligo sense + antisense Run a bit of each sample on a 2% agarose gel with ethidium bromide. For specific trademark information, see www.idtdna.com/trademarks. 27 0 obj<>stream they were designed to generate the overhang at the ends for cloning purpose, may be same as you are doing. So you will always end up with some degree of ssDNA contamination in your prep. *RUOFor research use only. Prepare oligos for annealing by adding 1 ul of each oligo (100 M stock) to a final concentration of 0.2 M (0.2 pmol/l) using 1X NEBuffer r2.1*. Kyriakos Hassapis: yes, at leasteither the vector or the oligos need to be phosphorylated for ligation to happen. Slow cooling to room temperature should take 4560 minutes. For annealing I use a buffer which has final 10 mM Tris pH 7.4, 1 mM MgCl2, 100 mM NaCl. All specimens will be evaluated at Mayo Clinic Laboratories for test suitability. If annealing was successful, the double-stranded band will be shifted up from the single-stranded band. Fatal error: Atomtype opls_116 not found Although I've already added this line: ; include water #include "oplsaa.ff/spc.itp" to [molecultype] directive in my topology. ,7903w=pVn 9V2JXf2um:.X9y'TKiN8yU%.,cR\o ZX"@ R3V @@bQH8?.L>L`AYC1cV@ + fc` T#7@ a Save time and money by placing an order with NEB. I am on the way to attempt extracting mitochondrial and nuclear DNA from some rather old, dry insect Hi all. Or, perhaps youd like guidance on which products are compatible? )v+5MyAF_Eg:.X l0ttt@J@ 2 0 obj I think the price per Oligo was $35 extra (try it on your own and you know how much work it is and how much you are paid to estimate if it is worth it). I have population genetic diversity data (mtDNA) and am trying to use the software PERMUT (http://www.pierroton.inra.fr/genetics/labo/Software/Permut) as described theoretically by Pons How to calculate the RMSD values for a MD simulation using MOE? 0000005407 00000 n Unsure of what products are available? https://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/annealing-oligos.html, 520 Waterfront Tech Center Annealing Oligonucleotides Protocol - Sigma-Aldrich Hi Michelle, I'm not sure about this but maybe it can work: plan you oligos so that they include a restriction site (ideally at the center of the o If you decide to go with the short-overlapping-oligo system which I can really recommend (use max 50 nt and you should be fine without HPLC and PAGE) then phosphorylate the oligos prior to annealing. If Normal Reports | In the end the problem was solved by designing small primers to anneal on the big oligos and thus amplify the whole region of 80 bp. Screening for selected oligosaccharidosis. Integrated DNA Technologies acquires Archer next generation sequencing research assays to advance actionable scientific discoveries. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), Alpha-N-Acetylgalactosaminidase Deficiency. No, I only ran the ssDNA (Forward/reverse) in water versus the dsDNA after the annealing reaction (the oligos were commercially synthetized and I need to anneal them to produce a fragment for cloning). endstream endobj 652 0 obj <>/Metadata 58 0 R/Outlines 95 0 R/PageLayout/OneColumn/Pages 649 0 R/StructTreeRoot 136 0 R/Type/Catalog>> endobj 653 0 obj <>/ExtGState<>/Font<>/XObject<>>>/Rotate 0/StructParents 0/Tabs/S/Type/Page>> endobj 654 0 obj <>stream This protocol uses a 1:50 (vector:insert) molar ratio with 0.02 picomoles of vector and 1 picomole of annealed oligos. If This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This page was printed from namlab.camden.rutgers.edu/protocols/annealing-oligonucleotides/ on Monday, May 1, 2023. cis-regulatory modules, sea urchin, and evolution, Center for Computational and Integrative Biology. Not for use in diagnostic procedures. WebPurify the fully digested plasmid Run out digestion product on gel There should be two bands: 7453 bp and 1935 bp Purify the larger 7453 bp band, not the 1935 bp stuffer band To save your cart and view previous orders, sign in to your NEB account. Fragments Entry. It makes sense what some of you said about the smear corresponding to unproper annealing. 1. Way more important is desalting for stability. Dispense 100 l aliquots of the mixed oligos into PCR tubes (500 l size). To obtain a long double stranded DNA with sticky ends, I started from multiple oligos (see attached file) instead of only 2 long oligos in order to avoid having internal annealing and secondary structure. Mix the oligos in PBS1X and put the tube in a water bath. Annealing should perform well over a wide range of oligo concentrations. You will find ds-oligos migrate differently from ss-oligos. 240 County Road gene fragments, Functional If the oligos have degraded, you should see a fuzzy band or a smear. Abnormal Reports, SI Normal Reports | WebAnneal oligos Re-suspend oligos in ddH20 (40 M). OLIGU - Overview: Oligosaccharide Screen, Random, Urine I saw the difference between 45 bases and 53 bases, but no difference between 53base oligo and annealed double-strand DNA. Copyright 2023 Rutgers, The State University of New Jersey. gRNA cloning McGraw-Hill; 2019. All Rights Reserved. xref If your plasmid or insert contain repetitive sequences, we recommend NEBStable CompetentE. coli(High Efficiency) (NEB #C3040H). For assistance, contact. Use different concentration of the annealed oligos in the ligation mix. You can proceed to cloning since only productive annealing will ligate. Briefly spin the tubes in a microfuge to draw all moisture from the lid. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners, and may be registered in the USA and/or other jurisdictions. You might be able to use a nanodrop. ssDNA has a higher absorbance by about 25% at room temperature. By comparing the absorbance of your annealed o What are the best ways to check for proper oligo Phenotype: continuum of clinical features ranging from severe and rapidly progressive disease to a milder and more slowly progressive course; clinical features of the early infantile type include fetal hydrops, edema, ascites, visceromegaly, dysostosis multiplex, coarse facies, and cherry red spot. Biochemical Genetics Patient Information (T602) in Special Instructions. Annealing Oligonucleotides Protocol | IDT - Integrated x[}Wo%Q%}m2E'9gxHiH}YM/-:*m]SNyeSMont_8oov\\_y_"g.Ruy70`z9U2a/fgcTo_#*%ReG6^2'$y.BaT~)}U7XUVsu^|tv\[1.w_[X./M^rzS"{~ggs+s2L+ P|xkhES7^^/.nusfZ.S];u%34asd+w3bs! }%0b13U[jy8)A v/-VW0f Vf6B _ 3p;@\woadUMbjk] t} 7tB3Y|g`uADFc] 88L&5Z] EM)r}lV?Zzxl.&@/q]f]jmW~cnr0YcqS8iU]=\l!_=Uz*@7qsZj~j 5. 0000029719 00000 n Can you tell me the procedure to anneal these oligos like how much should be the stock concentration while resuspending the oligos? All rights reserved. Camden NJ 08102 NEBuilder HiFi DNA Assembly Bundle for Large Fragments, Overlapping Oligos (100 M stock concentration), Annealed short DNA oligos forming a nicked dsDNA fragment, dsDNA vector linearized by PCR or restriction digest. This test may give false-negative results, especially in older patients with mild clinical presentations. You should run the annealing mixture at 2% agarose or better a native PAGE 5% acrylamide. WebProcess annealing, also called intermediate annealing, subcritical annealing, or in-process annealing, is a heat treatment cycle that restores some of the ductility to a product being cold-worked so it can be cold-worked further without breaking. Fragments Entry. Learn how we and our ad partner Google, collect and use data. Hi Michelle, here's the way we check it: Use 3 annealing reactions: 1) oligo sense only 2) oligo antisense only 3) oligo sense + antisense Run a bi Your product is now available from Integrated DNA Technologies. Is a hairpin in my oligo too strong for hybridization? | IDT endobj Available at: https://ommbid.mhmedical.com/content.aspx?bookid=2709§ionid=225545029, 3. Or, perhaps youd like guidance on which products are compatible? Oligo annealing protocol Resuspend after briefly spinning down each oligonucleotide pellet, dissolve in Duplex Buffer (100 mM potassium acetate; 30 If your oligos are above 50-60 nt the percentage of properly full length synthesized oligos will be decreasing rapidly. 0000004945 00000 n It is going to be difficult to distinguish between 80 bp annealed dsDNA product and an 80 base ssDNA on agarose gels. Just enter you sequence in the calculator Analysis module, and you'll find the T m reported in the seventh column. The smear could result from overloading. it worked nicely. %%EOF Does anyone know where I can find it? oligos Many of the Swift products you have grown to love are now part of our new complete portfolio, xGen NGS. 0000001563 00000 n The idea is to heat up the mixture to close to 100 C and let it slowly cool down to room temperature over the course of one hour. e`eXacOL>0h&8ljp=CtBfvpN>tck "?4[ > endobj 28 0 obj<> endobj 29 0 obj<>/ProcSet[/PDF/Text]/ExtGState<>>> endobj 30 0 obj<> endobj 31 0 obj<> endobj 32 0 obj<> endobj 33 0 obj<> endobj 34 0 obj<> endobj 35 0 obj<>stream @(o8Z-)*/]>b~tkrQxXW\a||[;IHV5TN^j8bgi z0=Oy. Oligosaccharide analysis may be considered in the workup of unexplained refractory epilepsy. 0000005291 00000 n use. better separation if you run on acrylamide gels and silver stain if the agarose Integrated DNA Technologies acquires Archer next generation sequencing research assays to advance actionable scientific discoveries. Are you doing COVID-19 related research? +(yT>d*Zbjow e#62i0SHWeTJ*mFp6F!Jkj1$"1/9LCChS7)F-?DVC!$3Kh'=O3&%VQnV? +:H.DHEMvE):Rrvnb~ YovW~3| P$/Y4jb5*5(tRPK5Y()xNiZ2 editing, Next 0000001426 00000 n Phenotype: normal appearing at birth followed by progressive neurodegeneration at 2-4 years, frequent respiratory infections, coarse features, thick calvarium, and osteoporosis. Transform a vector only ligation reaction. 0000001217 00000 n Hi, I want to start testing pitfall trap to obtain ants samples, but I need to conduct molecular analysis on those insects. I have done acconding to sigma annealing buffer composition (http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/annealing-oligos.html) and followed the protocol in waterbath (1.5 liter volume). Your product is now available from Integrated DNA Technologies. x\}W1x,`mG86p6`s*3HfI'R|OU%Nu+ju /_GyU>k\7(`5m?^n8BV~]L8)+e?~Z"i5Q;;XzQ stream 0000015415 00000 n 0000001297 00000 n WebHow do you calculate the annealing temperature of a primer? McGraw Hill; 2019. pPSTNz3w},`23wgw}_WKeo2 feF`Z]|uPtV^*Kd Fill out ourTechnical Support Form, If not ordering electronically, complete, print, and send a Biochemical Genetics Test Request (T798) with the specimen. Thomas GH:Disorders of glycoprotein degradation: alpha-mannosidosis, beta-mannosidosis, fucosidosis, and sialidosis. Thanks again for tying to help me out with this. stream The OligoEvaluator provides melting temperature for oligos. 0000015054 00000 n If you are looking at only annealing the oligos, any PCR buffer that claim to be good for high GC content PCR (e.g, Qiagen's kit with Q-solution) should be good enough. Otherwise as many people have suggested, incubating the tube in a container with water at 95 C and allowing it to sit at room temp until it cools down to RT should work equally well. Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Hope it works out for you. Heat the oligo mixture solution at 100C for 3 min and allow to cool at room temperature for 20 min. Phenotype: clinical features vary in severity and may include intellectual disability, respiratory infections, hearing loss, hypotonia, peripheral neuropathy, and behavioral issues. DD?Q~0dco.\K4K8`I0-](Nb]bJ_Uj!_.1}5}t~0 *. Ipswich, MA 01938-2723 Dispense 100 l aliquots of the mixed oligos into PCR tubes (500 l size). Available at: http://ommbid.mhmedical.com/content.aspx?bookid=2709§ionid=225544161, 2. I also agree with Anna. Neufeld EF, Muenzer J:The mucopolysaccharidoses. Oligosaccharidoses are characterized by the abnormal accumulation of incompletely degraded oligosaccharides in cells and tissues and the corresponding increase of related free oligosaccharides in the urine. WebThe annealing process is divided into two main steps: 1) dissolution, and 2) annealing, either by heat block or thermocycler. 19952023 Mayo Foundation for Medical Education and Research. WebHow can I tell if my oligos successfully annealed? You can arrange these structures in order of decreasing melting temperature (T m ). Annealing (materials science The Online Metabolic and Molecular Bases of Inherited Disease. Age of onset ranges from early infancy to adult and can even present prenatally. Tool, gBlocks Gene The step-wise thermocycler method maybe be a bit too much here because holding each temperature for a minute will allow for non-specific annealing, which may be contributing to your smear. The hybridized oligos or DNA duplex can be run on a non-denaturing gel with appropriate molecular weight markers. Not for use in diagnostic procedures. Designing and Using Oligos - Lubio Please note that DNA oligos with 5 phosphates are not required. 0000000916 00000 n Did you try to run it under denatured conditions to compare structural effects? When annealed, the overlapping oligos will form a nicked dsDNA fragment with no gaps, and ssDNA vector overlaps at each end. 671 0 obj <>stream Note that the final concentration of the oligo mixture is 50 M. Clinical features of oligosaccharidoses often overlap; therefore, urine screening is an important tool in the initial workup for these disorders. Mucolipidosis III-alpha/-beta and III-gamma (pseudo-Hurler Polydystrophy), Early childhood, may live well into adulthood, N-acetylglucosaminyl-1-phosphotransferase deficiency causing secondary intracellular deficiency of multiple enzyme activities. Make sure you have your oligos not (!) Entry, PrimerQuest You could try denaturing them at 95C+ then directly putting onto ice before running on a gel. Thank you so much for all your comments, hope I can help you people in the future somehow. genomics, GMP, OEM & Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners, and may be registered in the USA and/or other jurisdictions. We then run the annealed oligos through polyacrylamide mini gels and isolate the highest (and strongest) band. information, Webinars & video Resuspending the Oligonucleotides: Resuspend both complementary oligonucleotides at the same molar concentration, using Annealing Buffer (see note above). The oligosaccharidoses (glycoproteinoses) are a subset of lysosomal storage disorders (LSD) caused by the deficiency of any one of the lysosomal enzymes involved in the degradation of complex oligosaccharide chains. international site. I assume that you are going to use the annealed product as an adaptor or a linker of some sort for downstream application. Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Info@neb.com. I used to prepare a linker/adaptor for my thesis work which worked perfectly for me. Filter using one or more categories to focus on specific topics, or use the search bar to perform a textsearch. Collection Container/Tube: Plastic, 10-mL urine tube, 1. Web1ul of annealed oligo pair (or water in a control reaction) 3ul of prepped open vector (~20ng) 2ul 10x ligase buffer (NEB) 13ul of H2O 1ul T4 ligase (NEB) -------- 20ul total Ligation proceeds at 16C for 3-4 hours. Protocol cloning of oligos for sgRNA or shRNA constructs 2019 Make sure the oligos are properly desalted, check with the company what protocol they use. This is what we do: Transfer to a 37C water bath and incubate 30 min. email or call1-800-NEB-LABS. In:Valle DL, Antonarakis S, Ballabio A, Beaudet AL, Mitchell GA,eds. Ethanol expires too early and I need Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic How do I check if my oligos have degraded? - GenScript Help I was looking for many forum and it's still cannot solve my problem. Center for Computational & Integrative Biology sheets, Supplier *RUOFor research use only. ) T15w5CW,9d[Yn0sf.5rg O|]O_-diAr-]&dg w6~>vux[fS:$T!k)[&nO#7>2TT. Slowly progressive mental decline into adulthood. WebDo your annealed oligos form a blunt end and an EcoRI complimentary end? For more information see: -Epilepsy: Unexplained Refractory and/or Familial Testing Algorithm. Incubate the reaction at 95C for 4 minutes. Biochemical Genetics Patient Information (T602) in Special Instructions is recommended. WebUsing free OligoAnalyzer software, part of the IDT SciTools programs, enter your oligonucleotide sequence and choose Hairpin. The software will generate a series of possible hairpin structures. tutorials, DNA Oligo The annealed pair of oligonucleotides is ready for use. Hi there, we regularly make deletion cassettes for BAC recombineering using long overlapping oligos (100nt). You shoud use agarose (at least 3%; 1% is quite inappropriate) or polyacrylamide gel; try denaturing conditions to assess any structural or degradative effect. 1 0 obj Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. sgRNA Oligo Annealing and Ligation - Addgene Try another if you keep having degradation like you describe. You can arrange these structures in order of decreasing melting temperature (Tm). Later onset forms (types II and III) are milder and observed as progressive neurologic disease and vertebral dysplasia. Prenatal (type III) Infancy (type I) Juvenile/Adult (type II). Store on ice or at 4 C until ready to use.An alternative procedure for annealing involves the use of a thermal cycler. Tool, gBlocks Gene You may do the annealing on a PCR block by heating the mixture to 95 C and cooling slowly @ 1C per minute to 25 C. I suggest thermal cycler in order to control the cooling rate. oligos 0 Incubate the reaction at 50C in a thermocycler for 60 min. endstream endobj 655 0 obj <>stream Phenotype: infantile onset is characterized by rapidly progressive neurodegeneration, exaggerated startle reflex, "cherry red spot". stream hbbd``b`*qY&@Bf. Since you have two different enzymes your vector self ligation background should be very low. Hi Michelle El Khoury , Actually, i have the same problem :( Can you tell me, wich the appropriate protocol to check the annealing of the oligos ? gene fragments, Functional endobj After permethylation, the tubes are centrifuged, and the supernatant removed from the sodium hydroxide pellet. See The suggestion copied from IDT website (https://www.idtdna.com/pages/support/faqs/how-can-i-tell-if-my-oligos-successfully-annealed-) below, ho I would be highly grateful for any help. Type II is milder and slower progressing with survival into adulthood. WebOligosaccharidoses are characterized by the abnormal accumulation of incompletely degraded oligosaccharides in cells and tissues and the corresponding increase of 0000062323 00000 n 0000014272 00000 n The >80bp smear is most likely primers primers not bound in the correct way. Take advantage of free shipping for any order totaling over $350. F0 &>$5{*&1B3! 25 31 Infantile onset (type I) is characterized by rapidly progressive intellectual disability, hepatosplenomegaly, and severe dysostosis multiplex. or it has to be cloned in some vector? <>>> Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Transformation/plating/picking colonies 2ul ligation reaction mix is added to 25ul competent cells (DH5). Drug target identification via CRISPR screening, PCR Allele Competitive Extension (PACE) genotyping, SYBR Green dye assay and PrimeTime probe assays, Alt-R Predesigned Cas9 crRNA Selection Tool, Library Concentration Conversion Calculator, Target Capture Probe Design & Ordering Tool, CRISPR genome you can run a gel after annealing along side with single stranded oligos. If so, try our xGen NGS Solutions Builder Tool today. WebOligo annealing protocol Resuspend dried oligos to a concentration of 100uM in 10mM Tris buffer pH 8.0 (Check the amount of DNA in the tube in nanomoles (nm) listed on the 4 0 obj Place the tubes in a thermal cycler and set up a program to perform the following profile: i. I usually resuspend the oligos in water at high concentration (I always resuspend 1 in 50l and the second in how much will give an equimolar concentration, I don't bother with the specific concentration as long as both oligos are the same) I then add 1l of each oligo to 48l of annealing buffer (100 mM potassium acetate,30 mM HEPES-KOH pH 7.4,2 mM Mg-acetate) and in a PCR machine 4 minutes at 95C,10 minutes at 70C and Slowly cool down to 4C.
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