Ordering
Item | Catalog # | Description | Quantity | Price (USD) | ||
---|---|---|---|---|---|---|
Plasmid | 18917 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
AAV1 | 18917-AAV1 | Viral service discontinued. | Discontinued | |||
AAV9 | 18917-AAV9 | Viral service discontinued. | Discontinued |
This material is available to academics and nonprofits only.
Backbone
- Vector backboneAAV2(Search Vector Database)
- Backbone sizew/o insert(bp)5340
- Vector typeAAV, Cre/Lox ; Adeno-Associated Virus
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature30°C
- Growth Strain(s)Stbl3
- Growth instructionsStbl2 E coli from InvitrogenGrow at 30 degrees in 2xYT media
- Copy numberLow Copy
Gene/Insert
- Gene/Insert nameChannelrhodopsin 2-tdtomato
- Alt nameChR2
- SpeciesChlamydomonas reinhardtii
- Insert Size (bp)2367
- Tag/ Fusion Protein
- tdtomato (C terminal on insert)
Cloning Information
- Cloning methodRestriction Enzyme
- 5′ cloning siteSpe1(destroyed during cloning)
- 3′ cloning siteSpe1(destroyed during cloning)
- 5′ sequencing primerCTGTGGCTGCGTGAAAGCCTTG (Common Sequencing Primers)
Resource Information
- Supplemental Documents
- SmaI digest
- Addgene Notes
- Addgene diagnostic digest 3
- Digest of 18917 with PvuII, Sma1, and SnaB1
- Addgene Diagnostic Digest
- A portion of this plasmid was derived from a plasmid made byKarel Svoboda, Janelia Farm Research Campus
- Terms and Licenses
- UBMTA
- genOway Notice of RIghts
- Takara Bio Limited Use Label License (formerly Clontech)
- Industry Terms
- Not Available to Industry
- Articles Citing this Plasmid
- 29 References
Depositor Comments
These vectors are prone to recombination. This is a well known issue with these AAV vectors and is due to the inverted terminal repeats (ITRs) required for AAV production. To minimize recombination, we propagate these plasmids in Stbl2 cells from Invitrogen. Also, to minimize recombination, cells should be cultured at 30 C.
Note that these cultures will grow slowly (20 h for minipreps). Better yields and culture times are obtained with 2xYT as the media. This is strongly recommended.
Because recombination may still happen occasionally, we do a panel of restriction digestions to assess whether the ITRs are in tact. Separate digestions with PvuII, Sma1, and SnaB1 should be performed. The expected patterns can be calculated from the attached sequence. Please see Reviews in the right column for an image of Addgene's digest with these enzymes.
Information for AAV1 (Catalog # 18917-AAV1)(Back to top)
Addgene no longer distributes this item. Contact [email protected] for more information.
Viral service discontinued.
Purpose
Ready-to-use AAV1 particles produced from AAV-FLEX-rev-ChR2-tdtomato (#18917). In addition to the viral particles, you will also receive purified AAV-FLEX-rev-ChR2-tdtomato plasmid DNA.
CAG-driven, cre-dependent, channelrhodopsin fused to tdTomato for optogenetic activation.These AAV preparations are suitable purity for injection into animals.Delivery
- Volume.
- Titer≥ 1×10¹³ vg/mL
- Pricing$350 USD for preparation of . virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV1 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV1
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenetdTomato (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.
Information for AAV9 (Catalog # 18917-AAV9)(Back to top)
Addgene no longer distributes this item. Contact [email protected] for more information.
Viral service discontinued.
Purpose
Ready-to-use AAV9 particles produced from AAV-FLEX-rev-ChR2-tdtomato (#18917). In addition to the viral particles, you will also receive purified AAV-FLEX-rev-ChR2-tdtomato plasmid DNA.
CAG-driven, cre-dependent, channelrhodopsin fused to tdTomato for optogenetic activation.These AAV preparations are suitable purity for injection into animals.Delivery
- Volume.
- Titer≥ 1×10¹³ vg/mL
- Pricing$350 USD for preparation of . virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV9 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV9
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenetdTomato (Cre-dependent)
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
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例如:5000个核苷酸残基就是5000×0.15=750纳米
核苷酸(hé gān suān) Nucleotide,一类由嘌呤碱或嘧啶碱、核糖或脱氧核糖以及磷酸三种物质组成的化合物。又称核甙酸。戊糖与有机碱合成核苷,核苷与磷酸合成核苷酸,4种核苷酸组成核酸。核苷酸主要参与构成核酸,许多单核苷酸也具有多种重要的生物学功能,如与能量代谢有关的三磷酸腺苷(ATP)、脱氢辅酶等。
Musmusculus2dayspregnantadultfemaleovaryCDNA,RIKENfull-lengthenrichedlibrary,clone:E330021J16product:inferred:SRY-boxcontaininggene4/sox-4,fullinsertsequence。
product:inferred:SRY-boxcontaininggene4/sox-4,fullinsertsequence是什么意思,这个基因推测产物是sox-4?fullinsertsequence是什么意思?
这个缩写,N是nicotinamideA是腺嘌呤adenineD是二核苷酸dinucleotideP是磷酸基团phosphate那么H是什么的缩写呢?是还原H的意思吗?
上个封面先!
各位战友,你们好!我最近在做载体构建的实验,我打算将一段40bp左右的寡核苷酸连接到载体上,但是做了几次都是阴性结果,求各位大神指教!谢谢
我的操作过程如下:
我是将含有酶切位点的寡核苷酸引物稀释成100uM,各取1ul,加8ul水,95℃5mins,然后自然冷却至室温,然后再将退火后的核苷酸双链稀释了100倍。稀释后的核苷酸双链进行双酶切,酶切体系是:
10xBuffer4ul
Oligo30ul
酶12ul
酶22ul
ddH2O2ul
混匀后37℃水浴40min,在用65℃将酶变性20min
接下来就将寡核苷酸与双酶切回收载体连接(确认已经酶切开)
连接体系:
载体:2ul
oligo:10ul
10xT4buffer:2ul
T4连接酶:2ul
ddH2O:4ul
16℃连接过夜。
转化克隆后,挑取单克隆测序,发现全是没有连上,可能是什么原因呢?能帮忙解答一下吗?
以前听说过设计引物,以引物互为模板进行PCR获得的引物二聚体就是目的片段
有没有人具体操作过呢?应该注意什么问题呢?比如引物设计,体系,PCR程序
我冻干的5‘-核苷酸酶在固体状态挺稳定,但是复溶之后的液体状态分别在4℃和-20℃保存7天后,4℃保存的浓度下降20%以上,-20℃保存的没降。这是什么原因?该怎么解决?
核苷类药按其结构,也可分为3类: ① L-核苷类:,如拉米夫定和替比夫定;为嘧啶类②
无环磷酸盐类,阿德福韦酯和替诺夫韦;为嘌呤类,③ 环戊烷 /
烯类,如恩替卡韦。现在上市的有拉米夫定、替比夫定、阿德福韦和恩替卡韦;将要上市的还有替诺福韦。这一类新的药物还在不断研发,还会有更好的药物问世。
这一类药物有共同的特点。优点是应用方便,每天服一片药就行了;安全性强,很少不良反应,几乎没有禁忌症;最重要的是抑制病毒复制的活性很强,能较快改善病情。共同的缺点是需要长期用药,随意停药可能有反弹的风险;不计划、不规范用药容易发生耐药变异。
1、拉米夫定(贺普丁)有什么特点?
拉米夫定是第一个研发出来的口服抗病毒药,已经上市10年了,治疗了上百万人,救治了无数病人,取得了丰富的经验。
在上述几种口服抗病毒药中,拉米夫定抑制病毒复制的活性居于中等水平,一年平均可降低对数的5次方(即降到治疗前的1/10万)。随着病毒水平的降低,有七成病人血清转氨酶在3个月内降至正常;自觉症状也随之改善;肝组织病变在6个月内会有明显进步。肝硬化病人的恢复要慢一些。
拉米夫定是最安全的核苷类药,价格也较适当,过去、现在和在近期内都是应用最广泛的抗病毒药物,一个月的费用约460圆。
但拉米夫定也是最容易发生耐药变异的,每年以20%的比率递增。过去由于“一花独秀”,也有过一些误导,使我国有众多耐药变异的乙肝病毒感染者,因对其他核苷类药可能会发生交叉耐药,造成治疗困难。今后如能吸取教训,使用得当,仍不失为一个好药。
2、阿德福韦有什么特点?
阿德福韦抑制病毒复制的作用稍弱,1年降低4次方,故控制病情较缓慢,1年只有半数的病人血清转氨酶正常。但阿德福韦发生耐药变异较少,只要血清病毒长期维持在很低的复制水平,就有可能较长时间稳定控制病情。
阿德福韦是我国拥有自主知识产权的新药,目前国内有多家企业生产。在多种核苷类药物中,价格是最低的(一个月的费用约430圆),而且药品价格还在不断下调中。既能解决患者问题,又不至于增添患者太多经济负担。
但要注意的是:老年病人和肝移植病人可能有潜在的肾损害,用药中要多做检查。
3、恩替卡韦有什么特点?
恩替卡韦,在已经上市和尚未上市的几种药物中,抑制病毒复制的活性最强,一年平均可降低对数的7次方,几乎是拉米夫定的100倍,对极大多数病毒水平很高的病人也能在1年左右降至不能检出。早期作用更明显,头2周就能降低2次方,对救治重症病人特别有利。在肝移植病人可用于抑制病毒复现,可长期应用预防再感染导致的移植肝排斥。
恩替卡韦在没有用过核苷类药的初治病人,耐药变异的发生率很低,对已经拉米夫定耐药的病人疗效会明显降低。最大的问题是价格最贵,一个月的费用达1100圆。
4、替比夫定有什么特点?
替比夫定是拉米夫定的同类药,而抑制病毒作用约有拉米夫定的10倍,一年平均可降低血清病毒水平6次方,约60%的病人不能检出病毒。不过这些数据多来源国外,从临床疗效来看,控制病情、HBV-DNA阴转率、变异率稍优于拉米夫定,但从统计学上来分析,没有实际意义的差别。替比夫定一个月的费用约700圆,尚没有纳入国家医保。拉米夫定效果不佳时,可以考虑换用替比夫定,但一年后的耐药率也不低于10%。
怎样正确应用这一类药物?
因为服用这类药物很方便、很安全,有些病人像用止痛片或胃病药一样,随意停停用用,血清转氨酶升高了就用,正常了就停。这样不规范的用药很少有不出问题的,过去曾经有过不少教训。必须在医生指导下,按规定的剂量服药,要定期检查,发现问题要及时处理。
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