Available at: https://ommbid.mhmedical.com/content.aspx?bookid=2709§ionid=225545029, 3. endstream endobj startxref 0000034859 00000 n Clinical features can include bone abnormalities, coarse facial features, corneal cloudiness, organomegaly, muscle weakness, hypotonia, developmental delay, and ataxia. Kyriakos Hassapis: yes, at leasteither the vector or the oligos need to be phosphorylated for ligation to happen. Fax: 978-921-1350 tutorials, DNA Oligo you can increase the agarose to >2%, but I would just go to acrylamide gels instead. Transform a vector only ligation reaction. generation sequencing, Genes & $80012I20&` 0000054822 00000 n Valle DL, Antonarakis S, Ballabio A, Beaudet AL, Mitchell GA, The Online Metabolic and Molecular Bases of Inherited Disease, https://ommbid.mhmedical.com/content.aspx?bookid=2709§ionid=225545029, Epilepsy: Unexplained Refractory and/or Familial Testing Algorithm, Congenital Disorders of Glycosylation: Screening Algorithm, Congenital Disorders of Glycosylation (CDG, CDGN, OLIGU) Patient Information. *Note: you can also use TE buffer (10 mM Tris, 0.1 mM EDTA; pH 8.0) supplemented with 50 mM NaCl as an annealing buffer. Page last updated at 10:18 pm May 8, 2013. 0000002329 00000 n @OG,A)e\h.vzXCV}]7wvR+IRjNYF3a8397j_oc\wt+8Ix1}71l,7A(K*ga@;5V@87AD'JaazfT\.GW`TkFWq%aRZJ+Qg&|Uu&LI@-i3'4bT\jo)v:n:db!qx:lx%v TrAG5)rU>}$Ta>O(V+rJFRr43w)}WZf^= ,7903w=pVn 9V2JXf2um:.X9y'TKiN8yU%.,cR\o ZX"@ R3V @@bQH8?.L>L`AYC1cV@ + fc` T#7@ a For specific trademark information, see www.idtdna.com/trademarks. I think they only have to be 5-phosphorylated if the vector is dephosphorylated, right? & ,,r`E5nlE"kp>HIq_"] 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. 0000000016 00000 n McGraw Hill; 2019. You can use the PCR machine and a stepwise temperature program until you reach room temperature or even down to 4 `C.. Report back, After the annealing, when they have reach the room temp you can directly use for ligation with your linearized vector. WebAnnealing the phosphorylated FW and RV oligos: FW oligo RV oligo Sterile water Total 5 L 5 L 90 L 100 L Incubate the phosphorylated oligos at 95 0C for 3 minutes. )v+5MyAF_Eg:.X l0ttt@J@ If not ordering electronically, complete, print, and send a Biochemical Genetics Test Request (T798) with the specimen. You can arrange these structures in order of decreasing melting temperature (T m ). 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. Fragments Entry. oligos 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 Your product is now available from Integrated DNA Technologies. 0000005291 00000 n A nice, 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. ) T15w5CW,9d[Yn0sf.5rg O|]O_-diAr-]&dg w6~>vux[fS:$T!k)[&nO#7>2TT. Later onset forms (types II and III) are milder and observed as progressive neurologic disease and vertebral dysplasia. Integrated DNA Technologies acquires Archer next generation sequencing research assays to advance actionable scientific discoveries. Thank you so much for all your comments, hope I can help you people in the future somehow. WebYou can always put the gel back in the buffer and run longer if it has not gone far enough. Type II is adult onset characterized by angiokeratoma and mild cognitive impairment, and type III is an intermediate and variable form ranging from seizures and psychomotor delay to milder autistic features. After reading this thread, I got them PAGE purified. 0000001217 00000 n Learn how we and our ad partner Google, collect and use data. It makes sense what some of you said about the smear corresponding to unproper annealing. Way more important is desalting for stability. Phenotype: continuum within a wide spectrum of severity; clinical features include neurodegeneration, coarse facial features, growth delay, recurrent infections, dysostosis multiplex, angiokeratoma, and elevated sweat chloride. Tool, gBlocks Gene 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 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. better separation if you run on acrylamide gels and silver stain if the agarose Annealing Oligonucleotides | Nam Lab | Rutgers University 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. If the highest hairpin Tm is at or above your annealing temperature, that hairpin is likely to impede hybridization. 27 0 obj<>stream international site. Some companies do HPLC standard for desalting which works really well, I know Thermo Fisher does for their oligos, very good quality from my experience. 3/ I am not sure I have heard about any known chemistry that implicates salt in the degradation of DNA or RNA. Accessed August 4, 2021. Phenotype: continuum of clinical features ranging from severe and rapidly progressive disease to a milder and more slowly progressive course; infantile onset (type I) is characterized by rapidly progressive neurodegeneration. McGraw Hill Medical; 2009, Urine samples are extracted using Oasis HLB and carbograph columns and lyophilized overnight. 978-927-5054 Clients without access to Test Prices can contact, Prospective clients should contact their Regional Manager. % information, Webinars & video Slow cooling to room temperature should take 4560 minutes. Abnormal Reports, SI Normal Reports | Do not overlay the samples with oil. To determine if your oligos have degraded, we would recommend running them on a gel. Biochemical Genetics Patient Information (T602) in Special Instructions is recommended. ** NEBioCalculator (NEBioCalculator.neb.com) can help with DNA mass to molar quantity conversions for both ssDNA and dsDNA. All rights reserved. Many of the Swift products you have grown to love are now part of our new complete portfolio, xGen NGS. @(o8Z-)*/]>b~tkrQxXW\a||[;IHV5TN^j8bgi z0=Oy. integrations, User guides & Make sure the oligos are properly desalted, check with the company what protocol they use. you can run a gel after annealing along side with single stranded oligos. Age of onset ranges from early infancy to adult and can even present prenatally. use. 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 SI Abnormal Reports. Clinical features range from early developmental delay, coarse facial features, short stature, dysostosis multiplex, and hepatosplenomegaly to late onset cherry-red spot myoclonus syndrome. Oligo annealing protocol - iGEM Hi Ulrike, I wanted to also ask a similar question. 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 Entry, PrimerQuest Did you check for multiple complementary sequences within your oligo? << /Length 5 0 R /Filter /FlateDecode >> 2. Pool samples into a larger tube, store on ice or at 4 C until ready to use.Long Term Storage: It may be necessary to aliquot and lyophilize the annealed sample. My oligos are 63 bp long each. 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. endobj `d*]GAD`,B=C_XXJbo" I ;}A@rp]B];p+0!Ot#31TAeuXCU,)?Ri2inPP%z)NM c* `@=O0aoZV95D"\W9Wb$!v`L#Ass!9J'lPY$/&no,%D|"?cn#?'1Hp!r? Aq="5^cY~""* Hu6W61 `WR0n1`9R.&Vq~,`q(g6Iw6`LO3z;` p@)o?YT8W2\Va{/r=nNV0srTI4/M:XQ*6 (c24 1!kr1o)}_mfmPA`;8z(. Maybe you could post a gel image. 0000015415 00000 n Phenotype: infantile onset is characterized by prominent cardiomegaly, hepatomegaly, hypotonia, and weakness. The hybridized oligos or DNA duplex can be run on a non-denaturing gel with appropriate molecular weight markers. 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 2013 Sep;59[9]:1357-1368). <>>> Pseudo-Hurler polydystrophy is milder and later presenting. After permethylation, the tubes are centrifuged, and the supernatant removed from the sodium hydroxide pellet. p3&i\(Ojd3r1-u/d->zBI@X0. Learn about our tools that are helping researchers develop diagnostics and vaccines for the SARS-CoV-2 virus. For annealing I use a buffer which has final 10 mM Tris pH 7.4, 1 mM MgCl2, 100 mM NaCl. Seizures, hyperreflexia, and ataxia have been reported in more than 50% of later onset patients. (856) 225-6793 Proceed to a storage temperature of 4 C. Thanks again for tying to help me out with this. Use the same buffer and try to supplement the annealing buffer with 10% DMSO or formamide (at least 1M betaine should work as well). 0000002084 00000 n 0000018932 00000 n 0000029719 00000 n So you will always end up with some degree of ssDNA contamination in your prep. You could try denaturing them at 95C+ then directly putting onto ice before running on a gel. Use UV back shadowing or a stain such as SYBR Gold to visualize the bands. Neufeld EF, Muenzer J:The mucopolysaccharidoses. Tool, gBlocks Gene Your product is now available from Integrated DNA Technologies. For assistance, contact. 0000005407 00000 n I was looking for many forum and it's still cannot solve my problem. 4 0 obj (Toll Free) 1-800-632-5227 Annealing the Oligonucleotides:Mix equal volumes of both complementary oligos (at equimolar concentration) in a 1.5 ml microfuge tube. 0000014645 00000 n The number of colonies in this control should be <1% of the number of colonies in the uncut plasmid control transformation (from control #1). For more information see: -Epilepsy: Unexplained Refractory and/or Familial Testing Algorithm. You have to understand that annealing may not work at 100% efficiency. Type II is milder and slower progressing with survival into adulthood. startxref It worked! WebIf 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. % Since you have two different enzymes your vector self ligation background should be very low. Early infancy, late infancy or early adult, Cathepsin A causing secondary deficiencies in Beta-Gal and Neu. jn322@camden.rutgers.edu. % Center for Computational & Integrative Biology 2. Did you try to run it under denatured conditions to compare structural effects? gene fragments, Functional (Xia B, Asif G, Arthur L, et al: Oligosaccharide analysis in urine by MALDI-TOF mass spectrometry for the diagnosis of lysosomal storage diseases. Enzyme or molecular analysis is required to make a definitive diagnosis. I agree with Genevieve, though Taq polymerase a single cycle, 96C for 5 mins and 72 for 20 mins will ok as well. The annealed oligos are ready to assemble. %%EOF Boil for just 5 min and then take off from the heater. Milder later-adult onset forms of the disease exist presenting with neurological problems such as ataxia, dystonia, spinocerebellar degeneration, and behavior changes. Contact your local subsidiary or distributor. editing, Next I have an enquiry on statistical analysis. Contact your local US Sales Representative. Please sign back in to continue your session. The hybridized oligos or DNA duplex can be run on a non-denaturing gel with appropriate molecular weight markers. Phenotype: infantile onset is characterized by rapidly progressive neurodegeneration, exaggerated startle reflex, "cherry red spot". Camden NJ 08102 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). 0000018802 00000 n The plate is then analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF) 5800 Analyzer. How do I check if my oligos have degraded? - GenScript Help Our latest RUO kit, the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit, enables high throughput workflows for real-time detection of SARS-CoV-2 nucleic acid using hydrolysis probes. The OligoEvaluator provides melting temperature for oligos. McGraw-Hill; 2019. Phenotype: continuum of clinical features ranging from severe and rapidly progressive disease to a milder and more slowly progressive course; infantile onset (type I) is characterized by early developmental delay/arrest followed by progressive neurodegeneration, skeletal dysplasia, facial coarseness, hepatosplenomegaly, and macular cherry red spot. genomics, GMP, OEM & Make sure you have your oligos not (!) First, 1% agarose is not the right gel to see the difference between 80 bp DNA fragments. iAS;+o^mpa-]^]cH~]Vu}bkC/8z|[|"p__%C? Uj(Rl#`"a/-`BoWGb49w, Ev]8b(,.XL17&83ws/,VCM;ulb&Xf!zo?B]`M=+eE;bk Ww@(9j6_Rw I-uk0`> FoveEPjygXuW e=n,sa :/YN5EX$31`,x\@U[v']CbLx Z !GA &z]f|)\`ZrlS?;u(pDI LP| xb```e``~"620 Pcd0 mD. 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. protocols, Safety data This can be done by combining 1 l of each 100 M oligo stock in a single tube with an appropriate volume of buffer for a total volume of 500 l. 651 0 obj <> endobj Unsure of what products are available? 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. Although not always, some times in science the simplest method is the best method. I let them reach 25C before storing them at 4C. When I tried to energy minimization my system, I got fatal error as below. Hello everyone, thanks so much for helping. For larger scale oligo syntheses, it may be necessary to use larger volumes that can be aliquoted after resuspension. sgRNA Oligo Annealing and Ligation - Addgene 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. Annealing should perform well over a wide range of oligo concentrations. Oligo annealing protocol Resuspend after briefly spinning down each oligonucleotide pellet, dissolve in Duplex Buffer (100 mM potassium acetate; 30 If genomics, GMP, OEM & PrbbBB:Oligo Annealing it worked nicely. 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. Fragments Entry. 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. 3 0 obj Without seeing your sequence I can still assume such long DNA strands will likely pair up randomly as well as having various forms of self-hairpins. Filter using one or more categories to focus on specific topics, or use the search bar to perform a textsearch. Materials Thermocycler 10x annealing buffer, final (1x) concentration: 10mM Tris-HCl, 50mM NaCl closest match is NEB Buffer 2 (1x: 10mM TrisHCl, 50mM NaCl, 10mM MgCl 2, 1mM DTT) Procedure Hi there, we regularly make deletion cassettes for BAC recombineering using long overlapping oligos (100nt). 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. <<860137c47d3eb449aeb3c2531498d44f>]>> Many of the Swift products you have grown to love are now part of our new complete portfolio, xGen NGS. Rutgers University Save time and money by placing an order with NEB. Sorry, I didnt read all your thread. If you are getting smear.. then try to first heat the buffer til 70C and then add the oligos. 0000016683 00000 n 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. 0000015941 00000 n x[YoH~Ev,r}%$/[UTM.03#QU]WoT:s^8}viZeV&34'?^-b~{FU\x1:|T&[5*rtb o/GVW|-,euZ3MT@~P*f1Jf,SEY!_eYY8}g4+8#*#?#V ]V `Y8#2X"*&u+* i759r^d|*~K~= Integrated DNA Technologies acquires Archer next generation sequencing research assays to advance actionable scientific discoveries. 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. The Online Metabolic and Molecular Bases of Inherited Disease. 200 Federal St. %PDF-1.3 % Contact our Customer Service Team by 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. 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 Not for use in diagnostic procedures. I boiled the water on cooking heating plate and after boiling, I removed the container and kept aside withoud disturbing it. Some timeit is possible to obtain strange results running oligos on gels but the annealed oligos can be good enough for ligation. If you phosphorylate, then it is easy to check for annealing integrity by ligating the annealed product to itself so that you end up with 160 bp of ligated product versus 80 bp mostly ssDNA. OligoEvaluator for Tm Calculation & Primer Analysis were you have to solve this issue? I have the same problem 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 Earlier for congenital, infantile, and juvenile forms (type II). For convenience, keep Annealing Buffer volume below 500 l for each oligo. If you don't see your country above, please visit our This test may give false-negative results, especially in older patients with mild clinical presentations. If your oligos are above 50-60 nt the percentage of properly full length synthesized oligos will be decreasing rapidly. email or call1-800-NEB-LABS. Hope it works out for you. It allows seamless cloning and is fast, very efficient and cheap. generation sequencing, Genes & Resuspending the Oligonucleotides: Resuspend both complementary oligonucleotides at the same molar concentration, using Annealing Buffer (see note above). endobj 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). Annealing temperature should usually be several degrees below the T m value. The smear could result from overloading. Phenotype: continuum of clinical features ranging from severe disease (type II) to a milder and more slowly progressive course (type I). 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*. Normal Reports | <> Info@neb.com. Clinical features of oligosaccharidoses often overlap; therefore, urine screening is an important tool in the initial workup for these disorders. Almost all known nucleases have a zinc ion in their catalytic center. 671 0 obj <>stream oligos 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. My oligos have high Tmelting, hence using 97C. The Online Metabolic and Molecular Bases of Inherited Disease. oligos Use different concentration of the annealed oligos in the ligation mix. Hi, I want to start testing pitfall trap to obtain ants samples, but I need to conduct molecular analysis on those insects. %PDF-1.5 \N0I(29TO7j\'~~Ye_:G+Q@yqH#]iv":WWjw^oCC;Tz%g_i;2ey4{svN(rz*[+;pb BJL#@%c?Y?)Zu'A:?xPy\Ngn)[Sn ]Te-aX$B8NzLR\]\n@-Wkfe^L%08E~Pran\ZQYUy~-F1)dnU_D?;QhZI gRNA cloning If you repeat the annealing procedure, heat oligo mix in a thermal cycler up to 80C for 2-5 minutes to minimize degradative effects. You can arrange these structures in order of decreasing melting temperature (Tm). This protocol uses a 1:50 (vector:insert) molar ratio with 0.02 picomoles of vector and 1 picomole of annealed oligos. hbbd``b`*qY&@Bf. Prenatal (type III) Infancy (type I) Juvenile/Adult (type II). All Rights Reserved. 0000001563 00000 n 0 In:Valle DL, Antonarakis S, Ballabio A, Beaudet AL, Mitchell GA,eds. Protocol for Annealing Oligonucleotides (from Sigma-Aldrich)Annealing Buffer: 10 mM Tris, pH 7.58.0, 50 mM NaCl, 1 mM EDTANOTE: Oligos may also be resuspended in either 1x Ligase Buffer or 1x Kinase Buffer instead of the above Annealing Buffer (prior to annealing). Alternatively, run the duplexed oligo side by side with a single-stranded oligo. +(yT>d*Zbjow e#62i0SHWeTJ*mFp6F!Jkj1$"1/9LCChS7)F-?DVC!$3Kh'=O3&%VQnV? +:H.DHEMvE):Rrvnb~ YovW~3| P$/Y4jb5*5(tRPK5Y()xNiZ2 WebThe annealing process is divided into two main steps: 1) dissolution, and 2) annealing, either by heat block or thermocycler. Just enter you sequence in the calculator Analysis module, and you'll find the T m reported in the seventh column. Please note that DNA oligos with 5 phosphates are not required. $1rlEs hcRD(Sc=OVc]g*S3sD+mnamZzRi>OJYl5mgoa\'( 0000062323 00000 n WebWhen using oligos in PCR assays and panels for biologically related genes, avoid regions known to have a high rate of single nucleotide polymorphisms (SNPs) and span I am willing to share my protocol with you if you are interested. You may be seeing aggregates of "ssDNA" that run larger than the annealed sample. How can I tell if my oligos successfully annealed? | IDT This test may give false-positive results for Pompe disease, especially in pediatric patients on infant formula. !.q$!KiTDO5JjRAW&}1V0=qFq8q8Ait.qqop-OVEkPM.@, Hiiiii everyone! Thank you in advance. But the yield became less. Oligonucleotide phosphorylation and annealing - JBEI WebAnneal oligos Re-suspend oligos in ddH20 (40 M). Dispense 100 l aliquots of the mixed oligos into PCR tubes (500 l size). Designing and Using Oligos - Lubio You can proceed to cloning since only productive annealing will ligate. oligos 2. WebUpdated. %PDF-1.3 67m`#x30Dra#(([| ;rrnm|+8! Phenotype: clinical features vary in severity and may include intellectual disability, respiratory infections, hearing loss, hypotonia, peripheral neuropathy, and behavioral issues. Alternatively, run the duplexed oligo side by side with asingle-stranded oligo. Try digesting your vector with your two enzymes, gel purify (to get rid of the short vector sequence) and do not treat with CIAP. OLIGU - Overview: Oligosaccharide Screen, Random, Urine H\n@{?^&3;$d J ^RKX\RH~)7P~>M.W9Nx.P'/xu{~KEmlfuS0Uh1rN)eO6S^!}NLsne84i*RwY>Y>#3;33y\3k5ye"0 2gRhd. Heat the oligo mixture solution at 100C for 3 min and allow to cool at room temperature for 20 min. The annealed nicked dsDNA fragment can then be combined and assembled with a linearized vector fragment. Briefly spin the tubes in a microfuge to draw all moisture from the lid. The dss oligo will be brighter under the UV. The annealed pair of oligonucleotides is ready for use. Oligo Dissolution Although each oligonucleotide 0 editing, Next 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.

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