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Specifications
Print Version
| Description | SafeView? Classic is a new and safe nucleic acid stain for the visualization of nucleic acids in agarose and polyacrylamide gels. This dye eliminates the need for toxic Ethidium Bromide (EtBr, a potent mutagen), commonly used in gel electrophoresis. |
|---|---|
| SKU | G108 |
| SafeView Series | Gel Casting Dye |
| LED Viewer Compatibility | Yes |
| Stain Color | Green |
| Applications | Safe Detection of dsDNA, ssDNA and RNA in agarose and polyacrylamide gels. |
| Note | All SafeView DNA Stains are ISO-13485 certified. Dispose of SafeView DNA Stains as you would any other non-carcinogenic fluorescent dye (eg. Acridine orange; Propidium iodide). |
| Shipping Conditions | Shipped on blue ice packs. |
| Storage Condition | Store at 4°C for up to 2 years. |
| Unit quantity | 1.0 ml |
Documents
- Safe-Red Protocol
- Safe-Pack Protocol
- Safe-Green Protocol
- Safe-Red Protocol
- Safe-White Protocol
- SafeView DNA Stains Datasheet
- SafeView DNA Stains Troubleshooting Guide
- Safe-Green™
- Safe-Red™
- Safe-White™
- SafeView™ Classic
- Safe-Green™
- Safe-Red™
- Safe-White™
- G108 Stability Test- Re-Boiling Previously-solidified Agarose Gel
- Safe-Green™ Ligation Efficiency Test Report
- G108-series Visualization Product Testing Report
FAQs
| I want to know the differences of your products,Safe-Green,Safe-Red and Safe-white. | |
The main differences between Safe-Green, Safe-Red and Safe-white is their DNA binding dye and its excitation/emission spectra.Safe-Green has 500/522nm; Safe-White has approximate fluorescence excitation/emission maxima 358/461nm; Safe-Red has 488/562nm. Among these three, Safe-White has higher sensitivity than the other two colours. | |
| I have a question of usage for your Safe-White. As mentioned in your protocol,"Mix samples and DNA markers with a SafeViewTM dye at 1:5 dilution rate." means there is 50ul safe-white if the volume of my sample is 10ul? | |
If you have a 10ul sample, you should add 2ul of SafeView Dye to your sample before loading onto the gel. | |
| Can SafeView be a replacement for ethidium bromide? Can I do gel extraction with it? | |
SafeView can be used as a replacement for ethidium bromide as they both work on general agarose. We recommend using SafeGreen for downstream cloning applications as SafeView can interfere with the ligation reaction, yielding fewer colonies. | |
| How does SafeView work and why is it not carcinogenic? | |
There are fluorescent compounds in SafeView and these fluorescent compounds have the capability to bind to DNA. There may be some unknown effects of SafeView that have not been documented but that applies to the SYBR set as well; however, SafeView products are definitely not as carcinogenic as ethidium bromide. | |
| How do I use SafeView products? | |
The Safe-(Red, Green and White) loading dyes work the same as 6x loading dye, loaded with the sample. SafeView Classic is used directly in the gel and the running buffer. | |
| Does the SafeView differentiate double stranded nucleic acid and single stranded nucleic acid? Does the Safe-(Red, Green and White) work the same way? | |
Under UV, SafeView Classic emits a green fluorescence when bound to both single and double stranded DNA templates, therefore they cannot be differentiated by this method. It will emit a red fluorescence when bound to RNA templates.The SafeView Stains (Red, Green and White) do not perform in this way and will stain all nucleic acid templates one color. | |
| At what temperature do I store the SafeView products? | |
All the SafeView products should be stored at four degrees Celsius. | |
| Do I need a special filter for photography of DNA gels stained with SafeView? | |
Under UV light, SafeView Classic emits a green fluroescence when bound to both single and double stranded DNA templates. It will emit a red fluorescence when bound to RNA templates. No filter is necessary for viewing these colours, however a filter may be needed for photographing the gel. | |
| How long does the SafeView Classic stain last in a gel? | |
Our in-house testing has shown that SafeView stained gels (>10ng DNA loaded per lane) can still be effectively visualized up to 1 week later with only a slight decrease in brightness. Gels should be stored properly to maintain a good signal, at 4C in the dark, sealed in a plastic bag or pouch with wet paper towel loosely wrapped around the gel. | |
| Why is SafeView (G-108) stain not working on my samples? | |
Make sure you are following the protocol carefully. It is critical that both buffer and gel have SafeView dye in them otherwise it will not work. This is different from ethidium bromide. You can consider to add 2.5ul of SafeView (instead of 5ul) for every 100ml of running buffer, which will reduce background fluorescence and allow the bands to show with more contrast. | |
| Is it degradable, if so how fast and under what circumstances? | |
2 hours over 100C | |
| What is the sensitivity of the dyes? | |
Safe-Green has Excitation Wavelength of 490nm and Emission Wavelength of 525nm, and its sensitivity range is between 0.2-0.6ng.Safe-Red has Excitation Wavelength of 540nm and Emission Wavelength of 630nm, and its sensitivity range is between 0.3-0.8ng.Safe-White has Excitation Wavelength of 370nm and Emission Wavelength of 470nm, and its sensitivity range is between 0.2-0.5ng.SafeView Classic emits green fluorescence when bound to dsDNA and ssDNA and red fluorescence when bound to RNA. This stain has one excitation (490 nm) and two emission spectra (520 nm and 635 nm) and the sensitivity range is between 0.1-0.3ng.SafeView Plus has Excitation Wavelength of 490nm and Emission Wavelength of 525nm and its sensitivity range is between 0.05-0.1ng. | |
| Can SafeView products be used post-stain? | |
Only SafeView Plus (G468) should be used in a post stain. SafeView classic and Safe Stains are not designed for post-staining. SafeView Classic must be added to the gel and the running buffer prior to the loading of the samples. Safe-(Red, Green and White) stains must be added to the sample before loading it to the gel. | |
| Why is the EtBr signal stronger in the pictures when I compare SafeView with EtBr? | |
A reason for this is that most gel doc systems have been optimized for EtBr so that is why the EtBr signal may be stronger in the pictures. | |
| I have a question regarding the protocol. If I were to use Safe-Pack. All I need to do is mix my DNA sample with the Safe-pack loading dye? There is no need for additional components such as mixing the agarose gel with safeview classic and as well as mixing the running buffer. There is only one step and that one step is mixing the DNA sample with your dye, is this correct? | |
Yes, it is correct. Safe-Pack (Red, Green and White) has different different protocol from Safeview Claasic. Safe-Pack requires only to be mixed with DNA sample before loading the gel. There is no need to add dye in the gel or buffer. | |
| What is the functional pH range for SafeGreen? | |
SafeGreen works best between pH 7 to 9. | |
| Is there any guidance on how long the gel lasts in the fridge once the stain has been added? | |
We know that gels with Safeview can last a week in the refrigerator. If it is stored longer than a week, the agarose gel will have resolution problem. | |
| Is there any guidance on how long the gel lasts in the fridge once the stain has been added? | |
We know that gels with Safeview can last a week in the refrigerator. If it is stored longer than a week, the agarose gel will have resolution problem. | |
| How do I dispose Agarose Gels stained with Safeview and how do I dispose buffers and gloves? | |
Dispose SafeView™ Classic as you would any other non-carcinogenic fluorescent dye (eg. Acridine orange; Propidium iodide).All gels and contaminated “non-sharp” lab debris (e.g., gloves, pads, towels, tubes, etc.) that are processed using this dye can be discarded in the trash.Spent running buffer solutions and destaining solutions that contain the dyes can either be collected and disposed of through the HWMU or collected and run through an approved filter device.The buffer solutions that have been run through the approved filter should be checked under the appropriate light source for complete removal of the dyes, and if it passes (does not fluoresce), the liquid can be disposed of down the drain with a copious amount of water as long as no other materials are present that would cause the material to be a Hazardous Waste.The filters that have been used up and are no longer effective must be disposed of through the HWMU. | |
| will I need an additional loading buffer for my samples? | |
The loading dye is included in the products. No additional loading buffer needed. | |
| Do SafeView products give problems in the process of cloning? | |
We recommend using Safe-Green (G108-G) for downstream cloning applications, as testing has shown it yields more colonies following the ligation reaction than SafeView (G108). | |
| What is the difference between Safe Green, Safe Red and Safe White? Does the loading buffer have different colors? Which filters do I have to use to detect light emission? | |
The difference is simply in colour of the dye, when viewed under UV the bands in the gel will show up in green, red or white. | |
| We see migrations and band shifting of our fragments. Are there any recommendations that you can give us to minimize this band shifting? | |
Shifting is unavoidable and quite natural for any fragments, regardless of the staining agent. We suggest to use SafeGreen ladders, which will give accurate molecular weight with no additional staining agents needed.http://www.abmgood.com/DNA-Ladder-Safe-Green™-100bp-Opti-DNA-Marker-Invitroge-G473.htmlhttp://www.abmgood.com/DNA-Ladder-Safe-Green™-1kb-Opti-DNA-Marker-Invitroge-G474.html | |
| We see shifting and migration of the DNA fragments. What are the recommendations to minimize this? | |
Shifting is unavoidable and quite natural for such fragments, regardless of the staining. We suggest using SafeGreen ladders, which will give accurate molecular weight with no additional staining agents needed.http://www.abmgood.com/DNA-Ladder-Safe-Green™-100bp-Opti-DNA-Marker-Invitroge-G473.htmlhttp://www.abmgood.com/DNA-Ladder-Safe-Green™-1kb-Opti-DNA-Marker-Invitroge-G474.html | |
| I cannot see 100bp and 200bp bands on a 1% gel. What should I do? | |
It is very difficult to detect 100bp and 200bp bands in 1% gel with any stains. Higher gel concentrations should be used, such as 2% agarose. | |
| Can I use SafeView products in Polyacrylamide gels? | |
Yes, we have tested our SafeView products for this application. | |
| What device do you use to see the electrophoresis bands? | |
We use both UV and LED transilluminators, our gel QC pictures in PCR department are taken with UV. | |
| Will staining appear the same when using UV light and LED (same colors)? | |
UV and LED will give the same colors. | |
| Which of the Safe stains will work with blue light / LED? | |
All of our SafeView stains have been tested in-house to be compatible with UV light. SafeView Classic, SafeView Plus, and SafeGreen will also work under blue light/LED. SafeRed and SafeWhite will only work under UV light.Safe View Plus should only appear green. SafeView Classic will be red for RNA and green for DNA. | |
| Can this be stored at -20°C? | |
It should be fine to store at -20°C, it will only prolong the shelf life (normally 2 years). | |
| Can this be stored at -20°C? | |
No, please store at 4°C only. Shelf life of 2 years. | |
| What is the concentration of G108? | |
20000X (5ul of the G108 in 100ml gel solution). | |
| Are SafeGreen stained DNA bands suitable for ligation purposes? | |
Yes | |
| Does the dye migrate in a 1% agarose gel? | |
Yes, the dye should migrate on a 1% gel. | |
| Does G108-G contain SDS? | |
No. | |
| What is the concentration of G108-G? | |
6X loading dye | |
| Would adding Safe-Green to the DNA ladder interfere with the migration rate of the loading dye of the ladder? | |
No, adding SafeGreen to the DNA ladder will not interfere with the DNA ladder"s loading dye. You still need to add SafeGreen to the DNA ladder at the same ratio.To be more technically correct, SafeGreen, and all other EB alternative DNA binding dyes, would alter the migration pattern of DNA on agarose gel by shifting DNA to a larger molecular size; it is just simply because the overall molecular size of a particular DNA band increases as more dye is binding onto it. This is why we recommend mixing the DNA ladder and SafeGreen at the exact same 1:6 ratio, so everything on the gel will be subjected to the same concentration of SafeGreen and thus having the same shift. | |
| I"m a little bit confused about the light source that can be used for SafeRed. Below it is given, that the Ex/Em is 490/630nm, but in the last question (LED/UV light) it is given to use SafeRed with UV light source, normally in the range of 300nm. Is there a second excitation in the UV range? | |
Most labs are equipped with UV or LED light sources for viewing agarose gels, which is why we suggest these. As for SafeRed, yes, it does have an additional excitation wavelength of around 300nm. The other wavelength reported is for other people who may decide to use other light sources for the visualization. | |
| Can SafeView Classic penetrate cell membranes? | |
Yes, SafeView Classic can penetrate cell membranes. | |
| What is the size of the dye in SafeGreen after electrophoresis? | |
The dye should be at ~300bp after electrophoresis. | |
| After the electrophoresis runs, is it okay to recover the sample stained with Safe-red? Do I need to take special treatment to recover the sample DNA?I want to continue the cloning experiments with the sample after I check by electrophoresis. | |
Our Safe-Red™ (G108-R) will not affect the downstream cloning experiments, and thus no extra treatment is required to recover the stained sample DNA. | |
| Can Safe-Green penetrate the nucleus? | |
Yes, our Safe-Green™ stain indeed has the ability to penetrate the nucleus. | |
| Can this stain penetrate cell membranes? | |
Of our SafeView™ series DNA Stains, only SafeView™ (G108) and Safe-White™ (G108-W) can penetrate cell membranes, while the rest cannot. | |
| I used UV light for excitation, and my RNA is not glowing red as would be expected. Any idea why this is happening? | |
The complex interaction of SafeView Classic with DNA and RNA means that the emission spectrum is affected by secondary structure, temperature, ionic strength, pH and SafeView Classic concentration. Also, at high polynucleotides to SafeView Classic ratios, most nucleic acids will appear green. If the gels contain 6M urea at pH 3.5, all nucleic acids will appear green. So it is not unusual to observe the RNA bands as green under certain conditions. | |
| What color is the dye during electrophoresis? | |
The color of the dyes seen during migration would be blue, the same for all 3 dyes (Safe-Green/ Safe-Red/Safe-White). The size of the dye should be at approximately 300bp after electrophoresis. All three loading dyes have different excitation and emission wavelengths, so they will emit different colors. When bound to DNA, Safe-Green emits green fluorescence, Safe-Red emits red fluorescence, and Safe-White emits whitish pale blue fluorescence. | |
| What color is the dye during electrophoresis? | |
The color of the dyes seen during migration would be blue, the same for all 3 dyes (Safe-Green/ Safe-Red/Safe-White). The size of the dye should be at approximately 300bp after electrophoresis. All three loading dyes have different excitation and emission wavelengths, so they will emit different colors. When bound to DNA, Safe-Green emits green fluorescence, Safe-Red emits red fluorescence, and Safe-White emits whitish pale blue fluorescence. | |
| What color is the dye during electrophoresis? | |
The color of the dyes seen during migration would be blue, the same for all 3 dyes (Safe-Green/ Safe-Red/Safe-White). The size of the dye should be at approximately 300bp after electrophoresis. All three loading dyes have different excitation and emission wavelengths, so they will emit different colors. When bound to DNA, Safe-Green emits green fluorescence, Safe-Red emits red fluorescence, and Safe-White emits whitish pale blue fluorescence. | |
| Can you describe what particular imaging system that one should use to photograph the gel? | |
Any imaging system with UV and/or LED should work. No filter for Safeview is required, however a filter at ~500-650nm is optional. | |
References
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- Ashrafudoulla, M., Mizan, M. F. R., Park, H., Byun, K. H., Lee, N., Park, S. H., & Ha, S. D. "Genetic Relationship, Virulence Factors, Drug Resistance Profile and Biofilm Formation Ability of Vibrio parahaemolyticus Isolated From Mussel" Frontiers in Microbiology 10: (2019). DOI: 10.3389/fmicb.2019.00513.
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2015第四届国际分子诊断技术与应用论坛 查看更多
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2021-08-21
高圣医药受邀参加2015国际工程科技高端论坛中国分子诊断技术暨第六届中国分子诊断大会 2015国际工程科技高端论坛中国分子诊断技术暨第六届中国分子诊断大会于8月26-28日在中国贵阳召开。本次大会由中国工程院医药卫生学部主办发起，由清华大学、中国医药生物技术协会生物芯片分会、生物芯片北京国家工程研究中心等多家单位承办。本届会议精心准备，主要围绕遗传及出生缺陷分子诊断、遗传与分子诊断新技术、肿瘤精准诊疗、个体化医学检测管理与发展趋势等多... 查看更多
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2017-06-13
“2017(第五届)分子诊断产业高峰论坛”将于2017年6月19日－20日在杭州三立开元大酒店召开。2016年，国家精准医疗计划和个体化医学的发展要求持续推动了国内分子诊断技术的进步，进一步开拓新兴技术在临床的应用。液态活检作为精准医疗新技术，既有极高的科研价值，又具备助力推进精准医疗临床实践的巨大潜能，可用于癌症早期筛查、诊断、监控、治疗方案指导和疗效评估及产前诊断、器官移植诊断等领域。伴随诊断应用的发展，液态活检技术也逐渐被认可... 查看更多
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2021-09-03
最新分子诊断技术只需数小时快速诊断结核 查看更多
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2021-07-15
Dear Colleagues,
On behalf of the organizing commit 查看更多
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2017-05-14
医学分类对于建立诊断、预后以及选择治疗策略至关重要。传统的肿瘤分类主要基于解剖学和组织特点，现今，由于分子生物学和基因测序等技术的发展，肿瘤驱动基因的发现推动了肿瘤治疗由传统的放化疗向分子靶向治疗的转变。这一转变带来的是肿瘤分类学和治疗模式的转变，而基于分子分型的肿瘤分类使患者分层接受最优的靶向治疗，这就是精准医学的核心思路。肺癌是率先实现精准医疗的癌种。2004年起EGFR靶点的发现和EGFR-TKI药物的使用，推动了肺癌精准医疗的发 查看更多
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2021-09-21
长期以来，我国的临床分子诊断项目开展的科室主要是经过临检中心评审的医疗机构检验科，都基本建立了比较完善的实验室质量管理体系。检验人员一般都经过规范化培训，有较好的分子诊断相关检测操作质量意识。近年来以个体化治疗为特征的个体化精准医学正快歨走向临床医学的前台，带来了"药物基因组学"和"分子病理学"等概念。于是，近年来相关个体化医学检测在全国众多医疗机构的药学部、病理科、肿瘤科、妇产科、眼科，甚至中心实验室开展起来，状况与20世纪90年代初中期病原体核酸PCR检测情况类似。 查看更多
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2021-08-27
11月15日，分子诊断专题会议研讨会在中翰盛泰（杭州）生物科技产业园召开，中翰盛泰参与协办此次会议。来自全国各地分子诊断领域的顶级专家40余人应邀参与此次会议。 中翰盛泰董事长周旭一先生到会致欢迎词。，随后会议针对“实验室自建分子诊断项目（LDMTs）方法学确认专家共识”及“分子诊断规范报告”展开了积极而热烈的讨论。 会议休息期间，专家们参观了中翰盛泰展厅及园区其他功能区块，对我司有了进一步的认识和了解。 专题会议圆满结束后，专家们也... 查看更多
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2021-08-24
高圣医药首要参加2015（第三届）分子诊断产业高峰论坛！近年来，随着PCR，芯片，质谱等技术的发展和临床应用，中国分子诊断行业始终处于飞速发展状态。自2014年起，随着DNA测序成本的大幅降低、政府对高通量测序的叫停和开放、以及2015年初美国总统奥巴马在国情咨文演讲中宣布推出的“精准医疗计划”，人类基因组学、二代测序、计算机生物学分析等技术备受瞩目，更是促进了分子诊断行业的革新。 精准医疗的核心是基因科学，从科研产业化和技术转化的视野... 查看更多
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2021-06-03
靶向化药物和个性化治疗的发展和应用，是当代医学发展的主要潮流和方向，尤其是在恶性肿瘤、自身免疫、心脑血 查看更多
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2017-07-23
分子诊断是指应用分子生物学方法检测患者体内遗传物质的结构或表达水平的变化而做出诊断的技术。分子诊断是预测诊断的主要方法，既可以进行个体遗传病的诊断，也可以进行产前诊断。分子诊断主要是指编码与疾病相关的各种结构蛋白、酶、抗原抗体、免疫活性分子基因的检测。... 查看更多
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2021-08-07
嘉美生物是一家主要从事Annexin V,信号转导抗体,重组人和动物蛋白,磷酸化抗体,各种动物ELISA试剂盒,生物实验服务等研发与销售的高科技生物公司,免费热线:400-698-4168。 查看更多
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ABI7500与伯乐CFX96的对比123
刘鹏KRrj52017-10-03
伯乐的不错，实验目前用的就是这块，产品质量可以
全自动微生物鉴定和药敏分析系统 123
宮平专用c9j2017-10-03
数字pcr和荧光pcr哪个更准确在定量PCR时，我们常常纠结一个问题，究竟是相对定量还是绝对定量呢？如今，你无需纠结了，因为数字PCR（digitalPCR）来了。尽管这两种技术有些类似，都是估计起始样品中的核酸量，但它们有一个重要的区别。定量PCR是依靠标准曲线或参照基因来测定核酸量，而数字PCR则让你能够直接数出DNA分子的个数，是对起始样品的绝对定量。因此特别适用于依靠Ct值不能很好分辨的应用领域：拷贝数变异、突变检测、基因相对表达研究（如等位基因不平衡表达）、二代测序结果验证、miRNA表达分析、单细胞基因表达分析等。
3D数字PCR为精准医疗带来哪些优势 – 手机爱问123
zxn雫1522021-07-31
PCR（Polymerase Chain Reaction）技术，即聚合酶连反应，是一种扩大和复制目的片段DNA的技术，其发现对于生命科学的发展具有重要的意义。而3D数字PCR到底是什么鬼？它其实是最新出现的一款PCR仪，其具有对目的DNA分子做出绝对定量的优点，进而提供无与伦比的准确性、灵敏度。原理3D数字PCR是基于纳米流体芯片进行检测，通过一次检测，将样品分到数千个单独的PCR，检测结果部分为阳性，部分为阴性，之后进行绝对分子数统计，不需做标曲。目前可检测到2万个0.8nL的液滴（样品），能够精确的进行靶位点进行绝对定量研究。应用前景基因表达的绝对定量应用该技术，实现对DNA进行精确绝对定量，精确度可通过复制次数实现。通过准确性的进行定量检测基因，有助于临床上在治疗癌症、病毒感染等疾病作参考。同时，其具有高通量，检测速度快，成本相对低等优点，为后期精准治疗奠定基础。罕见基因的检测对于临床上的罕见的基因进行检测，不依靠参考标准，节约样本与试剂，并具有一定的精确度。3D数字PCR为液体活检提供技术支持液体活检是一种非侵入性的检测肿瘤基因及临床治疗效果的一种方法，目前可能正在开展有关3D数字PCR在在液体活检中的疗效，为后期临床提供更多可靠的指导作用。最新报道称“无创伤性肺癌检测技术即将登陆中国”。随着基因检测技术，如类似3D数字PCR技术的发展，推动我们将进入了“DNA的应用商城”这样的新时代，如23 and me、华大基因等专业的检测服务机构，提供更加专业的基因检测结果，经基因报告解读师或遗传咨询师进行通俗的解读，让我们每一个人都能够了解自己的基因，拥有自己的“生命之书”，让我们更加健康的生活。精准医疗其实本质是应用生物学信息与大数据科学的交叉发展的新型医学概念与医疗模式。对于精准医疗，重点在于“精准”，不仅仅是简单的基因测序如此简单，更需要精准的诊断与精准的治疗。
关于数字PCR,这些干货请收好[心得点评]123
周艳ing2021-07-28
请问论坛有做数字PCR的吗？为什么用外周血检测突变时，有扩增的微滴才几百个啊？大家一般有效微滴是多少个呢？
微滴式数字PCR(ddPCR)技术及应用123
2017-10-03
伯乐生命ddPCR（即微滴式数字PCR）系统能够确定核酸分子的绝对数目，让科研人员更加精确地研究生物学。已经被广泛应用到医学、生物学等方面，如拷贝数变异、突变检测、单细胞基因表达分析等。并且已经在癌症分子标志物的发现、传染病研究、基因组结构变异分析、基因表达分析等领域展现了强大的优势。
数字PCR的前世今生:特点、优势和局限性 分析行业新闻123
Yachenduan2021-07-29
请教有没有人知道哪里可以做数字PCR(dd-PCR)的公司，急求！
跪谢！
【旧贴整理】花了2星期系统归纳关于PCR的各位老师旧帖,与大家分...123
yyangyingy2018-01-22
常规PCR一般注意点：
1）PCR模板是关键，引物可以优化。制备模板的时候，一定要注意核酸模板的纯度和量。纯度上要避免杂质和杂蛋白质的混入，同时还要注意添加模板的量，模板很理想的话，不用加太多，加的多了，混进去的杂质机会就多了。当模板的浓度过低,比如低于100个分子时,引物和模板之间就很难发生反应.引物容易自身进行反应形成二聚体.用boosterPCR,即开始几个cycles保持primer的低浓度,保证primer:template的molarratio在107~108.以确保开始扩增的准确性.然后boostePrimer的浓度到正常的水平
2)退火温度是从55度开始.根据情况配合以Mg离子浓度进行调整.有条件的可以做grADIentpcr.退火的时间在30-60S,时间短一些可以得到更好的效果.
3）首先是你的TAQ酶要在冰上操作，以免失活（Taq酶容易失活所以冬天最容易出结果），TAQ酶使用时最好分装。再就是你的引物以及DNTP和模板操作完后一定放进冰箱，注意不要污染，PCR完成后要及时从PCR仪取出（如果你的PCR仪没有保温功能），如果你放在外面时间较长可以放－20度冰箱（尤其片段比较小时）。TAQ酶一般比较稳定,按照一般的惯例,分子生物学的东西从转运到加样,全部要在冰上操作
要注意的是,DMSO,GLYCEROL等会抑制polymerase的活性,所以需要scouting出最适的浓度.可以加入一些nonionic试剂,如Tween,Nonid,Trition之类的反过来抑制SDS.还有proteinaseK也要除干净,不然会降解polymerase.
一些高保真没的效率要远远低于Taqpolymerase,所以可能需要的酶的量也要大一些.另外,一般的情况下,变性的温度可以使用90~92度,变性的时间也可以缩短,
4)Mg离子的作用主要是dNTP-Mg与核酸骨架相互作用并能影响Polymerase的活性，一般的情况下Mg的浓度在0.5-5mM之间调整，同样要记住的是在调整了dNTPs的浓度后要相应的调整Mg离子的浓度
PCR实验中出现的问题及对策：
一、非特异性条带
原因：1>引物特异性差2>模板、引物↑;3>酶过量;4>Mg离子浓度↑;5>Tm↓;6>循环次数太多;7>被基因组DNA污染
对策：1>重设引物;2>引物、模板↓;3>减少酶量;4>Mg浓度;5>Tm适当;6>减少循环次数7>重新处理样品
具体措施:
1、Primer浓度过高，建议以0.1uM间隔递减
2、酶量过多，建议0.5U间隔递减
3、循环次数过多，建议2个循环间隔递减
4、Anneal温度过低，以1度间隔递增。不迷信文献上所谓的退火温度。摸下梯度，范围应该是Tm-10和Tm+5。一般选Tm值低5度的温度开始摸条件，很难摸出时可以使用降落PCR，普通的9600PCR仪也可以做，只是程序麻烦些。对于20个核苷酸，G+C含量约50%的引物，55℃为选择最适退火温度的起点较为理想。
5、采用Hotstart法或Coolstart法，减少从室温上升到变性温度过程中引起的Primer非特异性Annealing。简易热启动的方法是加样时最好在冰上加，当然可以做个简易冰盒，直接在94度放入样本到机器，效果不错。
6、Template量过多，Template量以20%递减。PCR模板是关键，引物可以优化。制备模板的时候，一定要注意核酸模板的纯度和量。纯度上要避免杂质和杂蛋白质的混入，同时还要注意添加模板的量，模板很理想的话，不用加太多，加的多了，混进去的杂质机会就多了。
7、Extension时间过短，30s间隔递增。
二、带微弱
1.出现的现象：扩增带时有时无，出现时与国外文献中的照片相似，不出现是则看不到或者非常弱。
2.曾用的解决方法：换过PCR中所用的试剂。甚至想到引物之间可能有二聚体等影响结果（虽然文献中及自己的实验中有时能出现很好的结果，但实在是万般无奈），把各引物单独或者两两结合进行试验，时好是坏。
3.思考：第一点，是否是管子的问题？管子的质量不是很好（为了便宜），特别是外形不是很规矩，与扩增仪上的管孔结合不紧密，又不经常加油，而原装管子则与管孔结合较紧密。是否由于此而使管内温度与实际设定温度不符呢？第二点，那么是否是变性温度较高使酶过早的失活？虽然扩增的产物很弱，但分子量是相符的，说明扩增在进行，只是产物量较少，这可能与Taq酶的活性有关.
4.最后解决：将变性温度由文献的95℃改为94℃，问题解决。以后各次扩增均得到满意的结果。
三、设立对照
设立阴性对照检测是否被基因组DNA污染。如果阴性对照的PCR结果也显示同样条带，则需要用重新处理样品。
设立阳性对照可以检测是否模板CDNA及引物有问题。
空白对照：加的是“水”——出现“区带”
待检样品：加的是“模板”——无“带”
说明：1，水被污染
2，模板阴性
四、遇到意外继续实验
PCR体系遇到停电就放在4度1天，还可以扩出来，意外时不要扔还可以做一次啊
PCR操作注意点
1、写好试剂标签
1）对于需要存放的试剂、样品，都有明确的标签，套小的密实袋，离心管上有品名、浓度、日期，在密实袋上会重复写一次；
2）实验记录上还会记下新来试剂的存放日期；
3）用成品试剂盒时，除了会在实验记录上记下某日用量多少，还会在试剂盒盖内侧标明某日用量。
2、管子放置
做大批量PCR用八连管，八连管的盖子有一端有缺口，有缺口的那段向着自己，有时碰到盖好后没有拿稳掉下来，也能准确无误的分清楚前后次序。
3、准备工作
实验之前，试剂器皿仪器都准备妥当，且在脑海中将实验目的和步骤联系起来再进行一遍对比检查，确认无误后，就快速准确的动手实验。
1）酶和dNTP最好分装．
dNTP的质量与浓度: dNTP的质量与浓度和PCR扩增效率有密切关系，dNTP粉呈颗粒状，如保存不当易变性失去生物学活性。dNTP溶液呈酸性，使用时应配成高浓度后，以1MNaOH或1MTris。HCL的缓冲液将其PH调节到7.0～7.5，小量分装，-20℃冰冻保存。多次冻融会使dNTP降解。在PCR反应中，dNTP应为50～200umol/L，尤其是注意4种dNTP的浓度要相等(等摩尔配制)，如其中任何一种浓度不同于其它几种时(偏高或偏低)，就会引起错配。浓度过低又会降低PCR产物的产量。dNTP能与Mg2+结合，使游离的Mg2+浓度降低。
2）PCR前，将枪等物品放在超净台中，用紫外照射半小时，可以破坏DNA，防止气溶胶污染等。但是，PCR不一定非要在超净台进行，如做质粒PCR就不那么严格．
3）PCR时，试剂化冻后，都稍稍振荡（手弹或者颠倒混匀），离心后再取（通常几秒钟，当转速到2000转左右关离心机）。（1）较均匀；（2）盖上沾的试剂被离下，不浪费试剂和减少污染。质粒DNA在取用之前，混匀的时间要稍长些。
4）PCR反应体系需要在冰上进行配制，以免酶失活或者核酸降解，反应用的器皿和微量离心管都需要进行严格的灭菌。
5)，两个引物可以加在一起分装或单独分装都可以.
4、预热PCR仪
上样前,预热PCR仪,对于PFU酶比较关键，先设置好PCR的程序然后再去配体系，别配好体系才想起PCR仪还没开。
5、加样：
1》、加样顺序
1）按照buffer-dNTP-引物-模板-Taq酶－水这个顺序加,不易起泡.
2）或加样顺序倒没有特别要求，只是Taq酶要注意最后加，加完就放到冰箱里或通常在冰箱里面加酶。然后按体积从大到小地一一加入.
3）或珍贵的、易引起污染的物质先加。
比如1、如果样品DNA很稀少，很宝贵，避免DNA间的污染就要首先考虑：第一步先加水，第二步就紧接着加DNA，这样加相同DNA就可以不换TIPS，又不会在DNA中污染引物等其他东西。
比如2、如果用随机引物，缓冲体系和酶就要重点保护？？？，先加它们再加引物和模板。
比如3、如果用的酶有外切活性，如PyroBest，pfu，就必需把酶放到最后加，否则他们有可能把引物都折磨得面目全非。
2》、混合加样
若做多管的PCR，可先加一起，再用枪分取
比如要做n管，就把除模板之外的其他部分按照n+1份配好，然后再分装，如：
10xbuffer5ul
dNTP4ul
引物11ul
引物21ul
taq酶1ul
ddw34ul
模板2ul
共有9个模板，就把除模板外的其他成分乘10，即buffer50ul，dNTP40ul，引物110ul，引物210ul，taq酶10ul，ddw340ul，加到一个离心管中，加盖，用力振荡使混匀（这步是必要的，否则分到各管中的体系不均匀），稍稍离心一下，然后分装至9个PCR管中，每管48ul，再依次加入模板2ul，混匀，稍稍离心，上机
3》、保证把微量的反应物加进去
1）枪头的下端紧贴液面(不要伸进去太多)，若看见有一个小水滴掉进去，证明已经加进去。不过我不赞成紧贴液面加，可能加进空气。所有的试剂都加完后，要用枪混匀，然后用手动离心机离一下，使之没有气泡，不要用手剧烈震荡tube
2）枪头不能挨到液面(加在管壁上瞬时离心)，否则使某些物质的量减少，所以我们加样的时候都采取螺旋式加样的方法，也就是加样时沿着管壁螺旋式上升，等全部加完后离心混匀，如果有气泡，用手指轻轻弹几下，气泡就没了（有时ep管内有气泡，反正p时要加温，发现最后结果相差不大），而且确保液体都在管底。
3）在用单个0.2mlPCR薄壁管做反应时，加在管壁，而不是管底，在管壁形成一个小液滴，每个小液滴都孤立存在，用手弹管壁最后离心。在加大量的药品时,如果1ml枪头一下吸满了液体,在枪头提出容器时,枪头尖端可能会存留一滴液体,很容易造成污染和浪费.我的技巧是在枪头提出容器时,将枪头放平,这样枪头尖端会有一个气泡,从而阻止了液体滴的形成!
4》、避免漏加或重复加
1）事先列出清单，准备两个冰盒.将所有的试剂放在一个冰盒上。加完的转移到另一个冰盒,加样以固定的顺序加,这样不容易弄错。
2）少量加样时，可以把试剂分别先加到EP管壁，这样不仅可以确定是否加上了试剂，还可以通过目测比较各种试剂加样是否准确；
3）加样时，将所有的试剂放在冰盒上。没加的在一侧，加完的转移到另一侧。
4）将PCR试剂放在最右边的竖排，再将EP管从左边开始横排放，在每加一种试剂后一定要将EP管挪动一排，这样就可以清楚的区分哪些管加了
5）还有如没有太大影响的话，将加完的合上盖子，没加的盖子打开。
6）泡沫（软的那种）制作了个板子，把ep管放上去，在加样时用枪尖压下
5》、准确浓度：
1）试剂完全化后再加样，浓度准确。像镁离子，融化后最后用枪头再吹打完全混匀。
2）各种酶，用之前瞬时离心一下，如果买的大包装的，最好分装一下，以避免反复冻融。
3）试剂用之前瞬时离心一下.
6》、注意枪头和枪是否结合紧密，否则误差将会很大；
7》、枪头：枪头要买好的，不然你有好枪也没用。
1）PCR模板是关键，引物可以优化。制备模板的时候，一定要注意核酸模板的纯度和量。纯度上要避免杂质和杂蛋白质的混入，同时还要注意添加模板的量，模板很理想的话，不用加太多，加的多了，混进去的杂质机会就多了。当模板的浓度过低,比如低于100个分子时,引物和模板之间就很难发生反应.引物容易自身进行反应形成二聚体.用boosterPCR,即开始几个cycles保持primer的低浓度,保证primer:template的molarratio在107~108.以确保开始扩增的准确性.然后boostePrimer的浓度到正常的水平
2)退火温度是从55度开始.根据情况配合以Mg离子浓度进行调整.有条件的可以做grADIentpcr.退火的时间在30-60S,时间短一些可以得到更好的效果.
3）首先是你的TAQ酶要在冰上操作，以免失活（Taq酶容易失活所以冬天最容易出结果），TAQ酶使用时最好分装。再就是你的引物以及DNTP和模板操作完后一定放进冰箱，注意不要污染，PCR完成后要及时从PCR仪取出（如果你的PCR仪没有保温功能），如果你放在外面时间较长可以放－20度冰箱（尤其片段比较小时）。TAQ酶一般比较稳定,按照一般的惯例,分子生物学的东西从转运到加样,全部要在冰上操作
要注意的是,DMSO,GLYCEROL等会抑制polymerase的活性,所以需要scouting出最适的浓度.可以加入一些nonionic试剂,如Tween,Nonid,Trition之类的反过来抑制SDS.还有proteinaseK也要除干净,不然会降解polymerase.
一些高保真没的效率要远远低于Taqpolymerase,所以可能需要的酶的量也要大一些.另外,一般的情况下,变性的温度可以使用90~92度,变性的时间也可以缩短,
4)Mg离子的作用主要是dNTP-Mg与核酸骨架相互作用并能影响Polymerase的活性，一般的情况下Mg的浓度在0.5-5mM之间调整，同样要记住的是在调整了dNTPs的浓度后要相应的调整Mg离子的浓度
PCR实验中出现的问题及对策：
一、非特异性条带
原因：1>引物特异性差2>模板、引物↑;3>酶过量;4>Mg离子浓度↑;5>Tm↓;6>循环次数太多;7>被基因组DNA污染
对策：1>重设引物;2>引物、模板↓;3>减少酶量;4>Mg浓度;5>Tm适当;6>减少循环次数7>重新处理样品
具体措施:
1、Primer浓度过高，建议以0.1uM间隔递减
2、酶量过多，建议0.5U间隔递减
3、循环次数过多，建议2个循环间隔递减
4、Anneal温度过低，以1度间隔递增。不迷信文献上所谓的退火温度。摸下梯度，范围应该是Tm-10和Tm+5。一般选Tm值低5度的温度开始摸条件，很难摸出时可以使用降落PCR，普通的9600PCR仪也可以做，只是程序麻烦些。对于20个核苷酸，G+C含量约50%的引物，55℃为选择最适退火温度的起点较为理想。
5、采用Hotstart法或Coolstart法，减少从室温上升到变性温度过程中引起的Primer非特异性Annealing。简易热启动的方法是加样时最好在冰上加，当然可以做个简易冰盒，直接在94度放入样本到机器，效果不错。
6、Template量过多，Template量以20%递减。PCR模板是关键，引物可以优化。制备模板的时候，一定要注意核酸模板的纯度和量。纯度上要避免杂质和杂蛋白质的混入，同时还要注意添加模板的量，模板很理想的话，不用加太多，加的多了，混进去的杂质机会就多了。
7、Extension时间过短，30s间隔递增。
二、带微弱
1.出现的现象：扩增带时有时无，出现时与国外文献中的照片相似，不出现是则看不到或者非常弱。
2.曾用的解决方法：换过PCR中所用的试剂。甚至想到引物之间可能有二聚体等影响结果（虽然文献中及自己的实验中有时能出现很好的结果，但实在是万般无奈），把各引物单独或者两两结合进行试验，时好是坏。
3.思考：第一点，是否是管子的问题？管子的质量不是很好（为了便宜），特别是外形不是很规矩，与扩增仪上的管孔结合不紧密，又不经常加油，而原装管子则与管孔结合较紧密。是否由于此而使管内温度与实际设定温度不符呢？第二点，那么是否是变性温度较高使酶过早的失活？虽然扩增的产物很弱，但分子量是相符的，说明扩增在进行，只是产物量较少，这可能与Taq酶的活性有关.
4.最后解决：将变性温度由文献的95℃改为94℃，问题解决。以后各次扩增均得到满意的结果。
三、设立对照
设立阴性对照检测是否被基因组DNA污染。如果阴性对照的PCR结果也显示同样条带，则需要用重新处理样品。
设立阳性对照可以检测是否模板CDNA及引物有问题。
空白对照：加的是“水”——出现“区带”
待检样品：加的是“模板”——无“带”
说明：1，水被污染
2，模板阴性
四、遇到意外继续实验
PCR体系遇到停电就放在4度1天，还可以扩出来，意外时不要扔还可以做一次啊
PCR操作注意点
1、写好试剂标签
1）对于需要存放的试剂、样品，都有明确的标签，套小的密实袋，离心管上有品名、浓度、日期，在密实袋上会重复写一次；
2）实验记录上还会记下新来试剂的存放日期；
3）用成品试剂盒时，除了会在实验记录上记下某日用量多少，还会在试剂盒盖内侧标明某日用量。
2、管子放置
做大批量PCR用八连管，八连管的盖子有一端有缺口，有缺口的那段向着自己，有时碰到盖好后没有拿稳掉下来，也能准确无误的分清楚前后次序。
3、准备工作
实验之前，试剂器皿仪器都准备妥当，且在脑海中将实验目的和步骤联系起来再进行一遍对比检查，确认无误后，就快速准确的动手实验。
1）酶和dNTP最好分装．
dNTP的质量与浓度: dNTP的质量与浓度和PCR扩增效率有密切关系，dNTP粉呈颗粒状，如保存不当易变性失去生物学活性。dNTP溶液呈酸性，使用时应配成高浓度后，以1MNaOH或1MTris。HCL的缓冲液将其PH调节到7.0～7.5，小量分装，-20℃冰冻保存。多次冻融会使dNTP降解。在PCR反应中，dNTP应为50～200umol/L，尤其是注意4种dNTP的浓度要相等(等摩尔配制)，如其中任何一种浓度不同于其它几种时(偏高或偏低)，就会引起错配。浓度过低又会降低PCR产物的产量。dNTP能与Mg2+结合，使游离的Mg2+浓度降低。
2）PCR前，将枪等物品放在超净台中，用紫外照射半小时，可以破坏DNA，防止气溶胶污染等。但是，PCR不一定非要在超净台进行，如做质粒PCR就不那么严格．
3）PCR时，试剂化冻后，都稍稍振荡（手弹或者颠倒混匀），离心后再取（通常几秒钟，当转速到2000转左右关离心机）。（1）较均匀；（2）盖上沾的试剂被离下，不浪费试剂和减少污染。质粒DNA在取用之前，混匀的时间要稍长些。
4）PCR反应体系需要在冰上进行配制，以免酶失活或者核酸降解，反应用的器皿和微量离心管都需要进行严格的灭菌。
5)，两个引物可以加在一起分装或单独分装都可以.
4、预热PCR仪
上样前,预热PCR仪,对于PFU酶比较关键，先设置好PCR的程序然后再去配体系，别配好体系才想起PCR仪还没开。
5、加样：
1》、加样顺序
1）按照buffer-dNTP-引物-模板-Taq酶－水这个顺序加,不易起泡.
2）或加样顺序倒没有特别要求，只是Taq酶要注意最后加，加完就放到冰箱里或通常在冰箱里面加酶。然后按体积从大到小地一一加入.
3）或珍贵的、易引起污染的物质先加。
比如1、如果样品DNA很稀少，很宝贵，避免DNA间的污染就要首先考虑：第一步先加水，第二步就紧接着加DNA，这样加相同DNA就可以不换TIPS，又不会在DNA中污染引物等其他东西。
比如2、如果用随机引物，缓冲体系和酶就要重点保护？？？，先加它们再加引物和模板。
比如3、如果用的酶有外切活性，如PyroBest，pfu，就必需把酶放到最后加，否则他们有可能把引物都折磨得面目全非。
2》、混合加样
若做多管的PCR，可先加一起，再用枪分取
比如要做n管，就把除模板之外的其他部分按照n+1份配好，然后再分装，如：
10xbuffer5ul
dNTP4ul
引物11ul
引物21ul
taq酶1ul
ddw34ul
模板2ul
共有9个模板，就把除模板外的其他成分乘10，即buffer50ul，dNTP40ul，引物110ul，引物210ul，taq酶10ul，ddw340ul，加到一个离心管中，加盖，用力振荡使混匀（这步是必要的，否则分到各管中的体系不均匀），稍稍离心一下，然后分装至9个PCR管中，每管48ul，再依次加入模板2ul，混匀，稍稍离心，上机
3》、保证把微量的反应物加进去
1）枪头的下端紧贴液面(不要伸进去太多)，若看见有一个小水滴掉进去，证明已经加进去。不过我不赞成紧贴液面加，可能加进空气。所有的试剂都加完后，要用枪混匀，然后用手动离心机离一下，使之没有气泡，不要用手剧烈震荡tube
2）枪头不能挨到液面(加在管壁上瞬时离心)，否则使某些物质的量减少，所以我们加样的时候都采取螺旋式加样的方法，也就是加样时沿着管壁螺旋式上升，等全部加完后离心混匀，如果有气泡，用手指轻轻弹几下，气泡就没了（有时ep管内有气泡，反正p时要加温，发现最后结果相差不大），而且确保液体都在管底。
3）在用单个0.2mlPCR薄壁管做反应时，加在管壁，而不是管底，在管壁形成一个小液滴，每个小液滴都孤立存在，用手弹管壁最后离心。在加大量的药品时,如果1ml枪头一下吸满了液体,在枪头提出容器时,枪头尖端可能会存留一滴液体,很容易造成污染和浪费.我的技巧是在枪头提出容器时,将枪头放平,这样枪头尖端会有一个气泡,从而阻止了液体滴的形成!
4》、避免漏加或重复加
1）事先列出清单，准备两个冰盒.将所有的试剂放在一个冰盒上。加完的转移到另一个冰盒,加样以固定的顺序加,这样不容易弄错。
2）少量加样时，可以把试剂分别先加到EP管壁，这样不仅可以确定是否加上了试剂，还可以通过目测比较各种试剂加样是否准确；
3）加样时，将所有的试剂放在冰盒上。没加的在一侧，加完的转移到另一侧。
4）将PCR试剂放在最右边的竖排，再将EP管从左边开始横排放，在每加一种试剂后一定要将EP管挪动一排，这样就可以清楚的区分哪些管加了
5）还有如没有太大影响的话，将加完的合上盖子，没加的盖子打开。
6）泡沫（软的那种）制作了个板子，把ep管放上去，在加样时用枪尖压下
5》、准确浓度：
1）试剂完全化后再加样，浓度准确。像镁离子，融化后最后用枪头再吹打完全混匀。
2）各种酶，用之前瞬时离心一下，如果买的大包装的，最好分装一下，以避免反复冻融。
3）试剂用之前瞬时离心一下.
6》、注意枪头和枪是否结合紧密，否则误差将会很大；
7》、枪头：枪头要买好的，不然你有好枪也没用。
起始进样量20ngdna的情况下,数字pcr检测ctdna单个位点的检测灵敏...123
ayの0623e萨2017-10-03
数字PCR可以绝对定量，理论上可以检测单个拷贝的突变，但是实际上如果突变分子比例太低，你可能取不到。20ng人基因组大致拷贝数是5.8E6，目前看文献报道，可以检出万分之一的突变比例，至于十万分之一，估计有点难度
数字虚拟PCR (In Silico PCR) PCR技术讨论版论坛123
yunxiang2021-08-03
http://www.genome.ucsc.edu/cgi-bin/hgPcr
使用了一下，发现很有用
对于查找文献中引物的位置
只要输入正义引物和反义引物，选择物种
就能预测pcr结果
可惜不能预测rt-pcr（因为数据库里没有mrna的数据）
大家以为如何，怎么这版没人提起过呢
谁知道哪里能预测rt-pcr结果呢
使用了一下，发现很有用
对于查找文献中引物的位置
只要输入正义引物和反义引物，选择物种
就能预测pcr结果
可惜不能预测rt-pcr（因为数据库里没有mrna的数据）
大家以为如何，怎么这版没人提起过呢
谁知道哪里能预测rt-pcr结果呢
数字pcr分析出现no call是什么原因123
夏羽12452021-07-22
可能是不是机器没信号造成的啊，我看你这个情况很像，建议楼主查找下说明书，按照说明书来具体操作呢
数字pcr 的应用做snp 频率怎么弄123
大神809052021-08-05
有很多方面你需要考虑，最重要的根据基因多态性频率为指标估算需要总样本量，如果其发生数服从Poisson分布，就采用Poisson分布的公式；其次根据基因多态性对你研究的疾病结局的相对危险度为指标估算需要病例数，具体的计算采用W. James Gauderma。
数字pcr一次可以检测多少样本_问答详情_数字PCR系统_分子诊断_...123
刘鹏CItt32017-10-03
在定量PCR时，我们常常纠结一个问题，究竟是相对定量还是绝对定量呢？如今，你无需纠结了，因为数字PCR（digital PCR）来了。尽管这两种技术有些类似，都是估计起始样品中的核酸量，但它们有一个重要的区别。定量PCR是依靠标准曲线或参照基因来测定核酸量，而数字PCR则让你能够直接数出DNA分子的个数，是对起始样品的绝对定量。因此特别适用于依靠Ct值不能很好分辨的应用领域：拷贝数变异、突变检测、基因相对表达研究（如等位基因不平衡表达）、二代测序结果验证、miRNA表达分析、单细胞基因表达分析等。
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