我们推荐立即将Dynabeads磁珠-mRNA复合物或洗脱的mRNA用于RT-PCR的cDNA合成或其他下游应用。如果需要保存，我们推荐使用10 mM Tris-HCl缓冲液（pH 7.5）从磁珠上洗脱mRNA并冷冻保存（-80°C）。所有设备和样本都必须是无RNase的。
答案ID：: CN7945这个回答对您是否有帮助？
What is the composition of your T4 DNA ligase buffer found in SKU 15224-025 and related products? 产品常问问题
Our 5X Ligase Reaction Buffer composition is as follows: 250 mM Tris-HCl (pH 7.6), 50 mM MgCl2, 5 mM ATP (Na+ salt), 5 mM DTT, and 25% (w/v) polyethylene glycol 8000.
答案ID：: EC3525这个回答对您是否有帮助？
What is your recommended RNase digestion mixture with Thermo Scientific RNase A/T1 Mix for RNase protection assay? 产品常问问题
We recommend 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA (pH 7.5), 20 L of RNase A/T1 Mix per 1 mL of reaction mixture.
答案ID：: EC8841这个回答对您是否有帮助？
What is the composition of the Thermo Scientific IP Lysis Buffer (Cat. No. 87787)? 产品常问问题
The Thermo Scientific IP Lysis Buffer contains 25 mM Tris.HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 NP-40 and 5 glycerol. This formulation is optimized for compatibility with immunoprecipitation and pull-down assays and does not contain SDS.
答案ID：: EC13382这个回答对您是否有帮助？
The highly concentrated mRNA can be eluted at 65 to 80 degrees C (2 minutes) in the desired volume, typically 10 to 20 L or down to 5 L. If eluted in a small volume, take care to pipette off the mRNA containing supernatant as soon as the beads have migrated to the side of the tube facing the magnet. The temperature changes in such small volumes will quickly allow for re-annealing of the mRNA to the Dynabeads Oligo(dT)25 magnetic beads. The suggested elution buffer is 10 mM Tris-HCl, but DEPC-treated water may also be used. However, if the eluted mRNA should be stored, Tris-HCl is strongly recommended.
答案ID：: EC4770
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I just received my Invitrogen GeneArt TALs vectors. Can I store them lyophilized? 产品常问问题
We recommend that you resuspend the vector in 50 L of distilled water or 10 mM Tris-HCl (pH 8.0) and incubate for 1 hour at room temperature. Resuspend the vector DNA by gently pipetting up and down 5-10 times. Store the resuspended DNA at 20 C.
答案ID：: EC10214这个回答对您是否有帮助？
可通过碱处理或高温处理分离DNA双链。采取碱处理时，可使用0.1 M NaOH洗脱非生物素化链。通常情况下，该处理方法不会对磁珠产生任何影响。
•在NaOH处理前，在2X结合和洗涤缓冲液（10 mM Tris-HCl，pH 7.5，1 mM EDTA，2 M NaCl）中清洗Dynabeads/DNA复合物1次，除去上清液。高盐浓度将有助于减少电荷，从而使非特异性结合最小化。
•向Dynabeads/DNA复合物中加入新鲜配制（很重要）的0.1 M NaOH溶液，室温下旋转孵育2-3分钟（最多5分钟）。除去含有非生物素化链的上清液。
•再用0.1 M NaOH溶液清洗Dynabeads/DNA复合物1次，除去上清液。首次洗脱时，即可洗脱下大部分DNA。
除了碱处理，也可进行加热处理，在水中95°C加热5分钟即可。
为防止再退火，碱处理或高温处理后必须快速从磁珠上分离DNA，最好在冰上操作。加热会在一定程度（在水中约为7%）上引起生物素化DNA从链霉亲和素上解离。因此，我们通常更推荐采用碱处理法。
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请考虑以下可能原因：
• pH > 9：检查PCR扩增反应的pH值，并使用1 M Tris-HCl, pH 8的缓冲液进行调节。
• PCR产物过多（或过度稀释）：减少PCR产物的用量（或增加其浓度)
• PCR反应延伸不完全：确保PCR反应最后包含一个7到30分钟的延伸步骤。长的PCR产物将需要更长的延伸时间。
•克隆长插入片段（>1 kb）：请尝试以下一个或全部建议：提高插入片段用量。延长 TOPO克隆反应孵育时间。使用硅基DNA纯化系统（如PureLink系统）或者电洗脱法对插入片段进行凝胶纯化。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
•尽管你使用了Taq聚合酶但是PCR产物仍然没有足够的3´ A突出：提高最后的延伸时间以确保所有的3´末端被腺苷化。Taq聚合酶在模板链的A后面再添加一个A时的效率较低。Taq聚合酶在模板链的C后边添加一个A时的效率最高。您可能需要重新设计引物以使它们包含一个5´ G而非一个5´ T。
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We offer several Dynabeads products for mRNA isolation, all of which are based upon the Dynabeads Oligo dT beads. The principle for isolation is A:T base paring between an oligo dT sequence covalently coupled to the beads and the PolyA tail of eukaryotic mRNA.
Dynabeads mRNA Purification Kit for the isolation of mRNA from total RNA (Cat. No. 61006: 2 mL/10 isolations). In addition to Dynabeads Oligo(dT)25 magnetic beads, the kit contains Binding Buffer, Washing Buffer, and 10 mM Tris-HCl.
Dynabeads mRNA DIRECT kit for direct isolation of mRNA from crude extracts of animal tissue, plant tissue, and cells (Cat. No. 61011: 5 mL/20 isolations and Cat. No. 61012: 2 x 5 mL/40 isolations). In addition to Dynabeads Oligo(dT)25 magnetic beads, the kit contains Lysis/Binding Buffer, Washing Buffers, 10 mM Tris-HCl, Reconditioning Solution, and Storage Buffer.
Dynabeads mRNA DIRECT Micro Kit for isolation of mRNA from small samples (Cat. No. 61021: 2 mL/100 isolations). In addition to Dynabeads Oligo(dT)25 magnetic beads, the kit contains Lysis/Binding Buffer, Washing Buffers, 10 mM Tris-HCl.
Whether you choose the Dynabeads mRNA DIRECT Kit or Dynabeads mRNA DIRECT Micro Kit depends on your sample size. The micro kit is designed for mRNA isolation from small samples (e.g.,
答案ID：: EC6065
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使用PureLink HiPure Mini, Midi, 或Maxi Purification试剂盒可以提纯lambda 噬菌体DNA吗？ 产品常问问题
是的，请参阅以下详细步骤：
1.使用2 mL (mini)/10 mL (midi)/30 mL (maxi) EQ1缓冲液平衡纯化柱。
2.收集噬菌体裂解物（液体或固体裂解物）并确定准确体积。
3.根据体积，加入30 μL/100 μL/400 μL缓冲液X1至10 mL/50 mL/250 mL 噬菌体裂解物中并在37°C孵育30 min。
X1 = 100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10 mM EDTA, 20 mg/mL RNase A, 6 mg/mL DNase I
4.将第3步中的核酸酶消化产物和2 mL/ 10 mL/50 mL冰预冷的缓冲液X2混合，冰上孵育60min。
X2 = 3 M NaCl, 30% (w/v) polyethyleneglycol (PEG) 6000
5.在>10,000 x g离心10分钟以收集噬菌体颗粒。弃上清。
6.用1 mL (mini)/3 mL (midi)/9 mL (maxi)缓冲液X3重悬噬菌体颗粒。
X3 = 100 mM Tris-HCl (pH 8.0), 25 mM EDTA
7.向噬菌体悬液中加入1 mL (mini)/3 mL (midi)/9 mL (maxi)的缓冲液X4。上下颠倒几次充分混匀并在70°C孵育10 分钟 (mini)或20 分钟 (midi and maxi)以裂解噬菌体颗粒。
X4 = 4% (w/v) SDS
8.向裂解物中加入1 mL (mini)/3 mL (midi)/9 mL (maxi)缓冲液X5，颠倒混匀，在≥13,000 x g室温离心10分钟。收集上清，避免吸取太多颗粒，然后直接将其加入平衡后的纯化柱内（见第1步）。让裂解物依靠重力进入树脂内。
X5 = 3.0 M 乙酸钾 (用乙酸调节至pH 5.5)
9.用2 x 2.5 mL (mini), 2 x 10 mL (midi), 或1 x 60 mL (maxi)缓冲液W8洗柱。
10.用0.9 mL (mini)/5 mL (midi)/15 mL (maxi)缓冲液X6洗脱lambda DNA并加入平衡至室温的0.7倍体积异丙醇沉淀DNA。
11.在≥13,000 x g，4°C离心30分钟。因为lambda DNA很粘，所以如果使用的是固定转角离心机则DNA将扩散至整个试管壁。因此，我们推荐使用水平转子离心机（例如HB-4或HB-6 Sorvall离心机），或者，如果没有此类转子，使用二甲基二氯硅烷硅化的离心管（如Corex）。
离心结束后，用80%乙醇洗涤lambda DNA并短时放置使其干燥。然后将lambda DNA溶解于适当体积的TE或10 mM Tris缓冲液(pH 8.0)中。
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What is AmpliTaq Gold PCR Master Mix (Cat. No. 4318739) and what does it contain? 产品常问问题
The AmpliTaq Gold PCR Master Mix is a convenient reagent premix used to perform polymerase chain reaction (PCR) amplification of single or multiple DNA targets. This Master Mix includes all of the chemical components (except primers and template) necessary for PCR. AmpliTaq Gold PCR Master Mix concentration is 2X and comes in a volume of 5 mL.
2X AmpliTaq Gold PCR Master Mix is composed of:
Gold DNA Polymerase, 250 U (0.05 U/mL)
PCR Gold Buffer, 30 mM Tris-HCL, pH 8.05
100 mM KCl
dNTP (400 mM each)
5 mM MgCl2.
For more information on this reagent, please consult the AmpliTaq Gold PCR Master Mix Protocol.
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If I suspect my gDNA requires further purification when using an Axiom array, what is the recommended cleanup protocol? 产品常问问题
If gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used:
1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at -20 degrees C), to gDNA.
2. Vortex and incubate at -20 degrees C for 1 hr.
3. Centrifuge at 12,000 x g in a microcentrifuge at room temperature for 20 min.
4. Remove supernatant and wash pellet with 80 ethanol.
5. Centrifuge at 12,000 x g at room temperature for 5 min.
6. Remove the 80 ethanol and repeat the 80 ethanol wash one more time.
7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
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Western blots can be stripped by incubation in Restore Western Blot Stripping Buffer at 37 degrees C for 5-15 min. If more stringent conditions are required, Restore Plus Western Blot Stripping Buffer can be used. Alternatively, membranes can be incubated in a stripping buffer consisting of 100 mM BME, 2 SDS, 62.5 mM Tris-HCL, pH 6.7, at 50 degrees C for 30 min with agitation. Wash the blot 3 times for 10 min each in PBST at room temperature. To reprobe with your antibody, the blot will need to be blocked again for 1 hr at room temperature.
Another stripping buffer is 0.1 M glycine-HCl (pH 2.5-3.0), the same solution commonly used for elution in immunoaffinity protocols. This solution will dissociate most antibody:antigen interactions at room temperature or 37 degrees C, but for strong antibody:antigen recognition, a 2 hr incubation may be necessary.
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What procedures are recommended for the preparation of beta amyloid-containing samples? 产品常问问题
Following are procedures which researchers have followed for handling beta amyloid in tissue and cell samples:
Kienlen-Campard, P, S. Miolet, B. Tasiaux, and J.-N. Octave (2002) Intracellular amyloid beta 1-42, but not extracellular soluble amyloid beta peptides, induces neuronal apoptosis. J. Biol. Chem. 277(18):15666-15670. These authors performed formic acid extraction of whole cells. Neurons (approximately 107) were scraped in ice cold PBS. Cell pellets were solubilized in 300 microliters of 70% formic acid. Formic acid-solubilized cell pellets were cleared by centrifuging at 16,000 x g for 5 minutes at 4 degrees C, and the supernatants were centrifuged at 21,000 x g for 20 minutes at 4 degrees C. The supernatants were vacuum dried and the resulting pellet was resuspended in 1 mL of alkaline carbonate buffer (2% Na2CO3, 0.1 N NaOH) and centrifuged 16,000 x g for 3 minutes at 4 degrees C. Protein concentration was determined by the BCA assay. For immunoprecipitation, 800 microliters of the supernatant was neutralized with 800 microliters of 1 M Tris-HCl, pH 6.8, and diluted 1:3 in H2O containing 10% FCS, 0.5% Triton X-100, and 0.5% Nonident P-40 (final concentrations). Beta amyloid 1-40 and Beta amyloid 1-42 concentrations were determined by ELISA using 100 microliters of neutralized extract.
Fagan, A.M., M. Watson, M. Parsadanian, K.R. Bales, S.M. Paul, and D.M. Holtzman (2002) Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer"s disease. Neurobiol. Dis. 9(3):305-318. This paper discusses analyzing beta amyloid by ELISA. This paper has an interesting variation on the measurement of beta amyloid: they looked at soluble beta amyloid and also insoluble beta amyloid. Here are the key points of their protocol: For analysis of total beta amyloid levels, half of the hippocampus from each animal was Dounce homogenized in 5 M guandine (50 nM Tris-HCl, pH 8.0) and beta amyloid ELISA was performed as described previously (Johnson-Wood, et al. 1997). For analysis of soluble beta amyloid, the other half of the hippocampus was homogenized on ice in 400 microliters TBS (25 mM Tris-HCl, 150 mM NaCl, 3 mM EDTA, pH 7.4) containing protease inhibitors (20 micrograms/mL aprotinin, and 10 micrograms/mL leupeptin). Homogenates were spun at 125,000xg in polyallomer tubes in a Sorvall RP100 AT4-406 rotor for 1 hour at 4 degrees C and levels of beta amyloid total in the resultant supernatant (defines as soluble Abeta total) were obtained by beta amyloid ELISA. Percentage of soluble Abeta total was defined as the soluble (TBS-extractable) value divided by the total tissue (guanidine-extractable) value.
For Brain Tissue Homogenization, Prepare the Following Solutions: 5 M guanidine HCl 50 mM TrisHCl, pH 8.0. Reaction Buffer BSAT-DPBS (Dulbecco’s phosphate buffered saline with 5% BSA and 0.03% Tween-20, see formulation below) supplemented with 1x Protease Inhibitor Cocktail from Calbiochem (catalog code 539131; contains AEBSF, aprotinin, E64, EDTA, and leupeptin). BSAT-DPBS Formulation 0.2 g/L KCl 0.2 g/L KH2PO4 8.0 g/L NaCl 1.150 g/L Na2HPO4 5% BSA 0.03% Tween-20 to 1 L ultrapure water and adjust the pH to 7.4. Protocol: Determine the wet mass of the mouse hemibrain (100 mg) or a human brain sample in an Eppendorf tube (Fisher K749520-0000). Add 8 x mass of cold 5 M guanidineHCl / 50 mM Tris-HCl (Solution "A", above) to the tube by 50 - 100 µL aliquots and grind thoroughly with a hand-held motor (Fisher K749540-0000) after each addition. (Optional: transfer the homogenate from above to 1 mL Dounce homogenizer and homogenize thoroughly.) Mix the homogenate at room temperature for 3 - 4 hours. The sample is stable and can be freeze-thawed many times at this stage. Dilute the sample with cold Reaction Buffer (Solution "B"). Centrifuge (microfuge or Sorvall) at 16,000 x g for 20 minutes at 4°C. Save the supernatant for the assay.
答案ID：: EC5147这个回答对您是否有帮助？
How much COT-1 DNA should I use to suppress repetitive sequences during hybridization? 产品常问问题
For Southern blot hybridizations, add 50 g of COT-1 DNA (at 10 g/ L) to 50 L of 20X SSC, 25 L distilled water and 20 L of a solution containing 0.1 M NaCl, 0.1 M Tris-HCl (pH 7.4). 0.01 M EDTA, and 1% SDS to the probe for each 25 to 500 ng of probe. For in situ hybridizations, combine genomic probe with the proper amount of COT-1 DNA such that the final concentration of COT-1 DNA is 0.3 g/ L for cosmid, plasmid, and lambda probes; or, at 1 g/ L for Alu PCR probes. Ethanol precipitate and resuspend in a half-volume of 100% formamide. Add a half-volume of 20% dextran sulfate in 2X SSC (prewarmed to 75 degrees C) and mix well. Denature mix by heating to 75 degrees C for 5 min. Incubate at 37 degrees C for 5 to 15 min.
答案ID：: EC3144这个回答对您是否有帮助？
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一、特级类胎牛血清1、Hyclone北美特级胎牛血清
这款血清--Hyclone特级胎牛血清(DefinedFBS)，有较好的促细胞生长作用及产品稳定性。内毒素含量小于10EU/ml，血红蛋白含量小于10mg/dl。此血清可以广泛用于细胞培养，包含干细胞系的培养。
2、Gibco北美特级胎牛血清
Invitrogen公司在美国原装生产的特级胎牛血清，是100nm过滤3次，内毒素和血红蛋白含量都可以与Hyclone北美特级胎牛血清的相媲美。也可以用于培养大部分的干细胞系。
3、CorningCellgro澳洲特级胎牛血清（1x500mL）FBS35-076-CV
二、优等类胎牛血清
1、Hyclone加拿大优等胎牛血清
其中一款血清是Hyclone加拿大优等胎牛血清(Characterizend FBS)，血清采自加拿大，由Hyclone公司总部的员工采集加工，采集方法，加工过程，检定标准与美国原产优等胎牛血清完全一致，市场价格低于美国原产的。经过3次100nm过滤，内毒素含量小于25EU/ml，血红蛋白含量小于25mg
。此血清，在2005年以前，是很多实验室选择血清的首选，价格适中，质量过硬，应用细胞非常广泛。
2、Hyclone澳大利亚优等胎牛血清
基本与加拿大的优等血清是一样的，级别也非常相似，各种级别都有。
3、Gibco澳大利亚胎牛血清与Hyclone澳大利亚胎牛血清差不多，这一级别的血清，也能用于干细胞培养，可是，不像特级胎牛血清那么优秀。
三、普通进口胎牛血清
目前市场上还有一款Hyclone血清是南美胎牛血清(FBS)，此血清采自南美，在国内(兰州)过滤并测试检验，经三次100纳米过滤，根据中国商标法的规定标贴中文标签，内毒素含量小于等于10EU/ml,血红蛋白含量小于等于20mg/d。此血清广泛用于各种肿瘤细胞的培养，还没有资料记载能够用于干细胞，也听说有少数人用于干细胞培养的，不过，因为本身血红素比较高，所以，最好不要用于干细胞。
2021年4月3日内容提示: Volumetric ANALYSISVolumetric ANALYSIS IntroductionAnalytical Chemistry: determining the chemical composition of samples of matter. Qualitat...
What is your recommended RNase digestion mixture with Thermo Scientific RNase A/T1 Mix for RNase protection assay? 产品常问问题 答案 What is the composition of ...
