
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | ||
---|---|---|---|---|---|---|
Plasmid | 59170 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
AAV1 | 59170-AAV1 | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
AAV5 | 59170-AAV5 | Virus (100 µL at titer ≥ 5×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
AAV Retrograde | 59170-AAVrg | Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information | Add to Cart |
This material is available to academics and nonprofits only.
Backbone
- Vector backboneAAV(Search Vector Database)
- Backbone sizew/o insert(bp)4368
- Total vector size (bp)6078
- Vector typeMammalian Expression, AAV
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)NEB Stable
- Growth instructionsStbl3 at 30C (use carbenicillin if using Stbl3) OR DH5a at 37C (use ampicillin if using DH5a)
- Copy numberLow Copy
Gene/Insert
- Gene/Insert nameChronos-GFP
- Alt nameChR90-GFP
- Alt nameStigeoclonium helveticum channelrhodopsin
- SpeciesStigeoclonium helveticum
- Insert Size (bp)1710
- GenBank IDKF992040.1
- PromoterSyn
- Tag/ Fusion Protein
- GFP (C terminal on insert)
Cloning Information
- Cloning methodRestriction Enzyme
- 5′ cloning siteBamHI(not destroyed)
- 3′ cloning siteEcoRI(not destroyed)
- 5′ sequencing primerCTGCGTATGAGTGCAAG
- 3′ sequencing primercagcgtatccacatagcg (Common Sequencing Primers)
Resource Information
- Terms and Licenses
- UBMTA
- Ancillary Agreement for Plasmids Containing FP Materials
- Industry Terms
- Not Available to Industry
- Articles Citing this Plasmid
- 3 References
Depositor Comments
Plasmid is completely sequenced by the depositing lab except for part of the 5' ITR.
Information for AAV1 (Catalog # 59170-AAV1)(Back to top)
Purpose
Ready-to-use AAV1 particles produced from pAAV-Syn-Chronos-GFP (#59170). In addition to the viral particles, you will also receive purified pAAV-Syn-Chronos-GFP plasmid DNA.
Syn-driven Chronos-GFP expression for optogenetic neural activation. 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
- Reporter GeneGFP
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.
Information for AAV5 (Catalog # 59170-AAV5)(Back to top)
Purpose
Ready-to-use AAV5 particles produced from pAAV-Syn-Chronos-GFP (#59170). In addition to the viral particles, you will also receive purified pAAV-Syn-Chronos-GFP plasmid DNA.
Syn-driven Chronos-GFP expression for optogenetic neural activation. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 5×10¹² 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 + 200 mM NaCl
- SerotypeAAV5
- PurificationIodixanol gradient ultracentrifugation
- Reporter GeneGFP
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
- 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.
Information for AAV Retrograde (Catalog # 59170-AAVrg)(Back to top)
Purpose
Ready-to-use AAV Retrograde particles produced from pAAV-Syn-Chronos-GFP (#59170). In addition to the viral particles, you will also receive purified pAAV-Syn-Chronos-GFP plasmid DNA.
Chronos-GFP under the control of the Synapsin promoter. These AAV were produced with a retrograde serotype, which permits retrograde access to projection neurons. 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, AAV retrograde cap gene from rAAV2-retro helper (plasmid #81070)
- BufferPBS + 0.001% Pluronic F-68 + 200 mM NaCl
- SerotypeAAV retrograde (AAVrg)
- PurificationIodixanol gradient ultracentrifugation
- Reporter GeneGFP
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
- 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
Retrograde functionality is dependent on high viral titers. Addgene recommends not diluting your AAV preps prior to use.ebiomall.com






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从大肠杆菌细胞中分离质粒DNA的方法众多,其分离的依据可利用分子大小不同,碱基组成的差异以及质粒DNA的超螺旋共价闭合环状结构的特点来进行。碱基性法抽提效果良好,既经济且得率较高。抽提到的质量DNA可用于酶切、连接和转化。对于分子量较大拷贝较少对的质粒DNA,由于DNA片段较大易于损伤断裂,因此选用吕华铯密度超高离心法抽提DNA,且具有纯度高、步骤少、方法稳定且获得的质量DNA是超螺旋构型等特点。对于高拷贝数质粒,用少量制备法抽提质粒DNA就有足够量可用于基因操作。
质粒DNA纯化的试验步骤:
测量DNA溶液的体积,按lg/ml的用量精确地加入固体CsCl, 将溶液加温至30℃助溶。温和地混匀溶液直到盐溶解。
每10mlDNA溶液加入0.8ml溴化乙锭溶液(10mg/ml溶于水);立即将溴化乙锭溶液(漂浮在表层)与DNA-氯化铯溶液混匀, 溶液的终密度应为1.55g/ml(溶液的折射率为1.3860)溴化乙锭浓度大约740μg/ml。【溴化乙锭贮存液应贮存于避光容器内(如用锡箔完全包裹的瓶子),于室温保存。
室温下用Sorvall SS34头(或与其相当的转头)以8000rpm离心5min, 浮在溶液上面的水垢状浮渣是溴化乙锭和细菌蛋白所形成的复合物。
用巴斯德吸管或带大号针头的一次性注射器将浮渣下的清亮红色溶液转移到离心管中。用轻石蜡油加满管的其余部分并封口。
以20℃对所得的密度梯度以45 000rpm离心16h(VTi65 转头)、 以45 000rpm离心48h(Ti50转头)、以60 000rpm离心24h(Ti65转头)或者以60 000rpm离心24h(Ti70.1转头)。普通光照下,在梯中心可见两条DNA区带, 上部区带材料通常较少,由线状的细菌(染色体)DNA和带切口的环状质粒DNA组成:下部区带则由闭环质粒DNA组成。管底部深红色的沉淀是溴化乙锭RNA复合物,位于CsCl溶液和石蜡油之间的是蛋白质。Beckman Quick-Seal离心管中的CsCl-溴化乙锭梯度可容纳4mg 闭环质粒DNA而不至超负荷。如有更大量的质粒存在,将扩展为一条宽带,并与染色体DNA相重叠。这种问题只有在质粒复制达到极高水平时才会出现,只要将该质粒提取物分为2个梯度即可解决。如出现负荷,可收集整个DNA区高产水平时才会出现,只要将该质粒提取物分为2个梯度即可解决。如出现超负荷,可收集整个DNA区带,用CsCl溶液(ρ=1.58g/ml)将体积调到15ml,在两个离心管中再度离心,使DNA达到平衡。
收集DNA带。将21 号皮下注射针头插入管的顶端以使空气进入,为尽量减少污染的机会,首先用18号皮下注射针头按下述方法收集上部的区带(杂色体DNA):用乙醇小心擦拭管外壁以除去任何油脂,然后将一块Soctch胶带贴于管外壁。穿过Soctch胶带将18号皮下注身针头(其斜面向上)插入管中,以便使针头的斜面开口恰好位于染色体DNA区带之下并与该区带相平行。将粘稠状DNA收集到一次性使用的管内,用造型粘土块封住皮下注射针头的末端并将第2根针头留于原处。 穿过Soctch 胶带插入第3根皮下注射针头(18号),将下部的质粒DNA区带收集到玻璃或塑料管中。展开
检测荧光信号的步骤有误:一般SG法采用72℃延伸时采集,Taqman法则一般在退火结束时或延伸结束采集信号。
引物或探针降解:可通过PAGE电泳检测其完整性。
模板量不足:对未知浓度的样品应从系列稀释样本的最高浓度做起。
模板降解:避免样品制备中杂质的引入及反复冻融的情况。
2.Ct值出现过晚(Ct>38)
扩增效率低:反应条件不够优化。设计更好的引物或探针;改用三步法进行反应;适当降低退火温度;增加镁离子浓度等。
PCR各种反应成分的降解或加样量的不足。
PCR产物太长:一般采用80-150bp的产物长度。
3.标准曲线线性关系不佳
加样存在误差:使得标准品不呈梯度。
标准品出现降解:应避免标准品反复冻融,或重新制备并稀释标准品。
引物或探针不佳:重新设计更好的引物和探针。
模板中存在抑制物,或模板浓度过高
4.负对照有信号
引物设计不够优化:应避免引物二聚体和发夹结构的出现。
引物浓度不佳:重庆新桥医院网上预约挂号www.cqxqyy.net适当降低引物的浓度,并注意上下游引物的浓度配比。
镁离子浓度过高:适当降低镁离子浓度,或选择更合适的mix试剂盒。
模板有基因组的污染:RNA提取过程中避免基因组DNA的引入,或通过引物设计避免非特异扩增。
5.溶解曲线不止一个主峰
引物设计不够优化:应避免引物二聚体和发夹结构的出现。
引物浓度不佳:适当降低引物的浓度,并注意上下游引物的浓度配比。
镁离子浓度过高:适当降低镁离子浓度,或选择更合适的mix试剂盒。
模板有基因组的污染:RNA提取过程中避免基因组DNA的引入,或通过引物设计避免非特异扩增。
6.扩增效率低
反应试剂中部分成分特别是荧光染料降解。
反应条件不够优化:可适当降低退火温度或改为三步扩增法。
反应体系中有PCR反应抑制物:一般是加入模板时所引入,应先把模板适度稀释,再加入反应体系中,减少抑制物的影响。
7.同一试剂在不同仪器上产生不同的曲线,如何判断?
判断标准:扩增效率,灵敏度,特异性
如果扩增效率在90%-110%,都是特异性扩增,都可以把数据用于分析。
8.扩增曲线的异常?比如“S”型曲线?
参比染料设定不正确(MasterMix不加参比染料时,选NONE)
模板的浓度太高或者降解
荧光染料的降解
这些染料的结构式基本是保密的。有些地方能查到,象维基上就有SYBR Green的结果式,但谁也不能肯定是否是正确的。
既说像坐标画曲线曲线应纵轴该荧光强度吧轴数值变化代表荧光强度变化荧光强度变化代表钙离浓度变化 我觉直接用荧光强度值变化代替钙离浓度变化研究意义该钙离浓度变化吧
非要荧光强度值换算钙离浓度需要做列工作
1 取标准钙离浓度染色用共聚焦显微镜测荧光强度
2 标准品需要同像环境主要曝光间相同激光波及能量
3 标准品要用同浓度荧光染料
才能根据标准荧光强度关系推算品钙离浓度
所以想请大家给推荐一个效果好的最好是国产的质粒提取试剂盒。
多谢!
对不同材料来说不同,绝大多数情况下,发射波长会随着激发波长的偏移而有所偏移。 对于固态物质,主要是因为分子与其它材料形成了π建 对于量子点溶液,激发波长也会显著导致发射光谱的不同。但是不是绝对的,比如对于Alex555分子,发射波长的便宜往往就相对较小,这是由于分子内部的能带结构所决定的。 如果是单纯的回答问题,答案是:有关。
你的荧光染料具体是哪一种?你可以通过染色试验来决定是否有效啊?一般荧光染料染色后,都会呈现明亮的颜色,比一般同样颜色要亮的多。只要能染上、只要有亮度,就是没失效。

