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Addgene/pAAV.CAG.Flex.GCaMP6m.WPRE.SV40/1unit/100839-AAV5
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Addgene/pAAV.CAG.Flex.GCaMP6m.WPRE.SV40/1unit/100839-AAV5
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Addgene
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100839-AAV5
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Ordering

ItemCatalog #DescriptionQuantityPrice (USD)
Plasmid100839Standard format: Plasmid sent in bacteria as agar stab1$75
Add to Cart
AAV1100839-AAV1Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information
$380
Add to Cart
AAV5100839-AAV5Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information
$380
Add to Cart
AAV9100839-AAV9Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information
$380
Add to Cart

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pAAV
    (Search Vector Database)
  • Vector type
    Mammalian Expression, AAV

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    NEB Stable
  • Copy number
    Unknown

Gene/Insert

  • Gene/Insert name
    GCaMP6m
  • Alt name
    GCaMP3 variant 629
  • Species
    Synthetic
  • Insert Size (bp)
    1353
  • Mutation
    GCaMP3-T302L R303P M378G K379S D380Y T381R S383T R392G
  • PromoterCAG

Cloning Information

  • Cloning methodUnknown
  • 5′ sequencing primerunknown
  • (Common Sequencing Primers)

Resource Information

  • Supplemental Documents
    • pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 ( p2817)_Q.gb
  • Terms and Licenses
    • UBMTA
    • genOway Notice of RIghts
  • Industry Terms
    • Not Available to Industry
  • Article Citing this Plasmid
    • 1 Reference

Depositor Comments

This plasmid was previously available as pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 ( p2817) from the Penn Vector Core. This plasmid was created as part of the GENIE project at Janelia Research Campus.

Information for AAV1 (Catalog # 100839-AAV1)(Back to top)

Purpose

Ready-to-use AAV1 particles produced from pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 (#100839). In addition to the viral particles, you will also receive purified pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 plasmid DNA.

CAG-driven, Cre-dependent GCaMP6m calcium sensor. These AAV preparations are suitable purity for injection into animals.

Delivery

  • Volume100 µL
  • Titer≥ 1×10¹³ vg/mL
  • Pricing$350 USD for preparation of 100 µL 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

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

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 AAV5 (Catalog # 100839-AAV5)(Back to top)

Purpose

Ready-to-use AAV5 particles produced from pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 (#100839). In addition to the viral particles, you will also receive purified pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 plasmid DNA.

CAG-driven, Cre-dependent GCaMP6m calcium sensor. These AAV preparations are suitable purity for injection into animals.

Delivery

  • Volume100 µL
  • Titer≥ 7×10¹² vg/mL
  • Pricing$350 USD for preparation of 100 µL 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, AAV5 cap gene
  • BufferPBS + 0.001% Pluronic F-68
  • SerotypeAAV5
  • PurificationIodixanol gradient ultracentrifugation

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
    • Terms of Use for Viral Vectors
  • Industry Terms
    • Not Available to Industry

Viral Quality Control

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 # 100839-AAV9)(Back to top)

Purpose

Ready-to-use AAV9 particles produced from pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 (#100839). In addition to the viral particles, you will also receive purified pAAV.CAG.Flex.GCaMP6m.WPRE.SV40 plasmid DNA.

CAG-driven, Cre-dependent GCaMP6m calcium sensor. These AAV preparations are suitable purity for injection into animals.

Delivery

  • Volume100 µL
  • Titer≥ 1×10¹³ vg/mL
  • Pricing$350 USD for preparation of 100 µL 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

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

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.

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最近有两位战友发信询问我的荧光微球吞噬实验的Protocol。

现分享于此:

LatexBeadsPhagocytosisassay

---Materials

*Fluorescencelabeledlatexbeads(1umdiameter),2.5%aqueoussUSPension
*3%BSAcontaining25mMNa2HPO4,pH6.0
*0,3%(w/v)azide
*Culturemediumcontaining5%FBS
*Distilledwater,PBS
*Bathsonication,6(12)-wellplates

---Cellcultureandtreatment

1,Inoculateplateswith7,0104cells/cm2perwell.Incubateat37℃,5%CO2for24hr,bestuntil50-70%confluenceisreached

2,Removeculturemediumandexposethecellstotestmaterial.Incubateat37℃,5%CO2for24hr.

---Preparationofcoatedlatexbeads

1,Washlatexbeadswithdistilledwaterandpelletat10,000gfor8minatRT.

2,Resuspendlatexbeadsin3%BSAcontaining25mMNa3PO4(pH6.0)andincubateatRTfor15minwithbathsonication.
*CoatingbeadsinBSAinsuresbeadsremaininamonodispersestate.

3,Washthebeadsoncewithculturemediumcontaining5%FBS.

4,Resuspendthebeadsinculturemediumatconcentration2.0%.Thisisbeadsstock.Storedindarknessat4℃.

---Assay

1,Controlsandsamples:Intactcontrol(Nostaining)1well
-Negativecontrol(azidetreated)1well
-Normalcontrol1well
-sample5wells

*Inordertodifferentiatebetweenphagocytosedbeadsandbeadsnonspecificallyadheretothecellsurface,controlcellsareexposedto0,3%(w/v)azidefor10minpriortotheadditionofcoatedbeads.Thistreatmentcompromisesmicroglialenergeticprocessesandfewbeadswereinternalizedasobservedbyfluorescentmicroscopy.Meanfluorescenceofazide-treatedmicrogliawasusedasthenegativecontrolandwassubtractedfromvaluesobtainedinexperimentalsamples.

2,Forexperimentsusing6well-plates,15μlbeadsstockin1mlculturemediumisappliedtoeachwell.Votexthebeadsstockwellandtakeout105μlandaddinto7mlculturemedium.Bathsonificatefor10minatRTindarkness.Thisisbeadsworkingsolution.

Forexperimentsusing12well-plates,6μlbeadsstockin0.4mlculturemediumisappliedtoeachwell.

3,WasheachwelltwicewithPBSandreplacewithbeadsworkingsolution,1ml/wellfor6-wellplate,0.4ml/wellfor12well-plate.Incubateinthedarkat37℃for80-120min.

4,Removebeadsworkingsolutionandwash3timeswithPBStoremoveexcessbeads.

5,LiftthecellsbyscrappingortrypsinizationandwashthecellswishPBS.

6,StainwithPI(4ug/mlfinalconcentration)andrunforFACS.
快到年关,才准备要做实时定量,但因从没做过,万一设计有误,那损失不堪设想,恳请各位高手帮我看看有没有什么问题:
我要检测的是一个巢氏PCR的终产物,那我能否用首次PCR的产物纯化,稀释后来作标准品?好象一般都认为用质粒作更好,但我想质粒片段与我样品首次PCR产物的长度不同,扩增效率是不是也有差异?而且用质粒扩增前,是不是要先把质粒酶切成线性后,才能扩?(这个问题好象有点菜)
另外,如果用荧光染料,那是用预混型试剂好,还是各配各的好?因为据说镁离子浓度对实验影响较大,如果用预混型,不是就不能调离子浓度了吗?

先谢谢各位了。
用不同荧光染料标记的抗体,分别与小鼠细胞和人细胞的细胞膜上的一种抗原结合,两类细胞则分别产生绿色荧光和红色荧光.将两类细胞融合成一个细胞时,其一半呈绿色,一半呈红色.在37℃下保温40min后,细胞上两种荧光点呈均匀分布(如图所示),试问:(1)人...
带荧光的染料含不含重金属
经常对对照品与标准品的概念不清楚,有何区别,查了一下,得到如下结果:
对照品系指用于鉴别、检查、含量测定和校正检定仪器性能的标准物质,而标准品系指用于生物检定、抗生素或生物药品中含量或效价测定的标准物质,以效价单位(U)表示。
还是感觉不甚明了,是否标准品只用于生物方面?是否化学方面只能称对照品?标准品有什么要求?对照品有什么要求?

From:http://www.cdda.gov.cn/index/zcfg_detail.asp?ID=62a
国家药品标准品、对照品系指国家药品标准中用于鉴别、检查、含量测定、杂质和有关物质检查等标准物质,它是国家药品标准不可分割的组成部分。国家药品标准物质是国家药品标准的物质基础,它是用来检查药品质量的一种特殊的专用量具;是测量药品质量的基准;也是做为校正测试仪器与方法的物质标准;在药品检验中,它是确定药品真伪优劣的对照,是控制药品质量必不可少的工具。
目前,中国药品生物制品检定所已能提供各类国家标准物质1242种,其中中药化学对照品288种,对照药材400种,两者占总数的一半以上,

from:http://www.chp.org.cn/2005gs/swfl/swfl.htm
国家标准品及生物参考品系指用于鉴别、检查含量或效价测定的标准物质,其制备与标定应符合“生物制品国家标准物质制备和标定规程”要求,并由国务院药品监督管理部门指定的机构分发。企业工作标准品或参考品必须经国家标准品或参考品标化后方能使用。
对照品系指用于生物制品理化等方面测定的特定物质,由生产单位采用与制品生产工艺相同的方法制备。对照品应尽可能与制品原液配方一致,稳定性较差的,可加不含对测定有干扰物质的适宜的稳定剂。对照品由国家药品检定机构审查认可,其标准应不低于制品的质量标准。

From:http://www.pharm.sdu.edu.cn/peiyang/jiaoan/yaowufenxihx.doc
(3)标准品、对照品:是指用于鉴别、检查、含量测定的标准物质,均由国务院药品监督管理部门指定的单位制备、标定和供应。标准品系指用于生物测定、抗生素或生化药品中含量或效价测定的标准物质,一国际标准品进行标定;对照品出另有规定外,按干燥进行计算后使用。
标准品和对照品均附有使用说明书,质量要求,有效期和装量等。

From:http://www.chp.org.cn/2005gs/swfl/swbz.htm
1定义
生物制品标准物质系指用于生物制品效价、活性或含量测定的或其特性鉴别、检查的生物标准品或生物参考物质。
2标准物质的种类
生物制品标准物质分为二类。
国家生物标准品系指用国际标准品标定的,或我国自行研制的(尚无国际生物标准品者)用于定量测定某一制品效价或毒性的标准物质,其生物活性以国际单位(IU)或以单位(U)表示。
国家生物参考品系指用国际参考品标定的,或我国自行研制的(尚无国际参考品者)用于微生物(或其产物)的定性鉴定或疾病诊断的生物试剂、生物材料或特异性抗血清;或指用于定量检测某些制品的生物效价的参考物质,如用于麻疹活疫苗滴度或类毒素絮状单位测定的参考品,其效价以特定活性单位表示,不以(IU)表示。

From:http://www.proteomics.com.cn/msl/papers/Notice1.html
1 对照品与标准品概念不清
对照品与标准品是2个不同的概念,中国药典凡例中已有明确的定义:对照品系指用于鉴别、检查、含量测定和校正检定仪器性能的标准物质,而标准品系指用于生物检定、抗生素或生物药品中含量或效价测定的标准物质,以效价单位(U)表示。文献中常将2种概念混淆,认为对照品就是标准品,是1种物质2种提法而已[1,2],造成错误的原因,可能是有的药品既有对照品,又有标准品。例如,当用微生物法测定头孢克罗效价时,用头孢克罗标准品,用HPLC或UV法测定时,则用对照品;非那西丁当用作熔点校准物质时,用熔点标准品,测定含量时,用对照品。即使是同一种物质的标准品和对照品,它们的规格、标定方法以及用途都可能是不同的。
2 对照品或标准品混用
对照品或标准品混用,即将对照品或标准品用于不是其标定方法的含量测定,是药品检验中经常出现但未引起重视的一个问题[3]。尽管同一批对照品不同标定方法的含量有很好的相关性,但并不完全相同,有时差别会很大。如英国Glaxo公司提供的头孢呋肟酯对照品,HPLC标定为96.9%,供含量测定用;UV为98.8%,供溶出度测定。虽然中国药典凡例明确规定卫生部所发对照品仅用于正文中所规定的分析方法。但由于:(1)卫生部提供的对照品使用说明书不够详尽,大多无对照品质量要求及标定方法;(2)对对照品或标准品的正确使用缺乏认识;(3)日常科研中极难找到相应的对照品;(4)中国药典正文中也常存在对照品混用的问题,如常将含量测定用的标准品或对照品用于溶出度检查,而含量测定方法与溶出度分析方法又不同,故极易引起混用。
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