Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | ||
|---|---|---|---|---|---|---|
| Plasmid | 100838 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
| AAV1 | 100838-AAV1 | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV9 | 100838-AAV9 | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
This material is available to academics and nonprofits only.
Backbone
- Vector backbonepAAV(Search Vector Database)
- Vector typeMammalian Expression, AAV
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)NEB Stable
- Copy numberUnknown
Gene/Insert
- Gene/Insert nameGCaMP6m
- Alt nameGCaMP3 variant 631
- SpeciesSynthetic
- Insert Size (bp)1353
- MutationGCaMP3-T302L R303P M378G K379S D380Y T381R S383T R392G
- PromoterSynapsin
Cloning Information
- Cloning methodUnknown
- 5′ sequencing primerunknown (Common Sequencing Primers)
Resource Information
- Supplemental Documents
- pAAV.Syn.Flex.GCaMP6m.WPRE.SV40 (p2820)_Q.gb
- Terms and Licenses
- UBMTA
- genOway Notice of RIghts
- Industry Terms
- Not Available to Industry
Depositor Comments
This plasmid was previously available as pAAV.Syn.Flex.GCaMP6m.WPRE.SV40 (p2820) from the Penn Vector Core. This plasmid was created as part of the GENIE project at Janelia Research Campus.
Information for AAV1 (Catalog # 100838-AAV1)(Back to top)
Purpose
Ready-to-use AAV1 particles produced from pAAV.Syn.Flex.GCaMP6m.WPRE.SV40 (#100838). In addition to the viral particles, you will also receive purified pAAV.Syn.Flex.GCaMP6m.WPRE.SV40 plasmid DNA.
Syn-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
- 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 # 100838-AAV9)(Back to top)
Purpose
Ready-to-use AAV9 particles produced from pAAV.Syn.Flex.GCaMP6m.WPRE.SV40 (#100838). In addition to the viral particles, you will also receive purified pAAV.Syn.Flex.GCaMP6m.WPRE.SV40 plasmid DNA.
Syn-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
- 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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1．目的
学会PCR操作的基本技术。
2．原理
是将待扩增的DNA模板加热变性，与其两侧互补的寡聚核苷酸引物复性，然后经过耐热的DNA聚合酶延伸。再进入下一轮变性—复性—延伸的循环，n次循环后DNA可被扩增（1+X）n倍。其中<25nt的引物退火温度Tm=2（A+T）+4（G+C）。
3．器材
旋涡混合器，微量移液取样器，移液器吸头，0.2mlPCR微量管，双面微量离心管架，PCR仪，台式离心机，琼脂糖凝胶电泳系统，水漂，恒温水浴。
4．试剂
5U/μlTaqDNA聚合酶，PCR缓冲液（10×，MgCl2free），25mMMgCl2，dNTP，引物，模板质粒pBS-CHI，无菌ddH2O。
5．实验准备
dNTP混合液（每种25mM），TAE电泳缓冲液，荧光染料，10´加样缓冲液，1.5ml离心管装入铝制饭盒（灭菌）、移液器吸头装入相应的吸头盒（灭菌）。合成的引物：Senseprimer5＇-GGATCCACAATGATGAGAGCC-３＇
Antisenseprimer５＇-GATATCATGGTGAGGTAGCTAGCTT-３＇
目的基因模板质粒：已经克隆在pBS载体上的1128bp几丁质酶（chitinase，CHI）基因。
待扩增的CHI片段长度：996bp。
6．操作步骤
（1）在0.2mlPCR微量离心管中配制50μl反应体系。（以下加样量供参考，括号内是最终需要量，实验时需参照Taq酶说明书计算）
ddH2O32μl
10×PCRbuffer（不含MgCl2）5μl
25mMMgCl23μl
2.5mmol/LdNTP4μl（每种dNTP终浓度0.2mM）
10μmol/LPrimer12μl（12.5~25pmoles）
10μmol/Lprimer22μl（12.5~25pmoles）
模板质粒1μl（1×10-3pmoles）
5U/μlTaq酶1μl（5U）
总体积50μl
（2）根据厂商的操作手册设置PCR仪的循环程序：
①94℃5min
②94℃1min
③54℃1min
④72℃1min50s
⑤goto②29times
⑥72℃10min
（3）PCR结束后，取10μl产物进行琼脂糖凝胶电泳。观察胶上是否有预计的主要产物带。
（4）清理桌面，撰写实验报告。
（5）PCR产物可以直接与T载体连接（见实验五），然后转化感受态细胞（见实验八）。
7．思考
（1）复性温度如何确定？
（2）为什么要在最后延伸10min？
（3）是否有非特异性扩增产物（或引物二聚体），如何才能消除？
更多资讯请关注于http://www.chinazglab.com/
这里所说的一个碱基的差异该怎么检测？如何理解？
为适应医疗器械监督管理工作的需要，总局组织有关单位和专家对恒温核酸扩增检测仪等22个产品的管理类别进行了界定。现通知如下：
一、作为Ⅲ类医疗器械管理的产品（1个）
恒温核酸扩增检测仪：由检测系统、加热模块、温控系统、触摸屏和随机软件组成。基于荧光检测的恒温核酸扩增检测技术，定性检测样本中的目标核酸有无扩增。分类编码：6840。
二、作为Ⅱ类医疗器械管理的产品（7个）
（一）免疫磁微粒捕获仪：由蠕动泵、管路、永磁铁及其他必要辅助器具组成。用于磁微粒免疫分析中捕获磁微粒复合物，是临床免疫磁微粒检验分析的前处理设备。分类编码：6841。
（二）精液采集器：由精液收集套、润滑剂、精液采集管、采集漏斗、袖珍剪组成。产品以无菌型式提供。临床上用于收集成年男性精液，为评价男性生育能力提供完整标本。分类编码：6841。
（三）干式血细胞微量样品测试管：由测试管体、封管器、浮子、肝素钠抗凝剂和荧光吖啶橙染料组成。用于收集末梢血样和静脉血样，临床上与干式血细胞计数仪配合使用，对样本染色、抗凝和分类。分类编码：6841。
（四）胎盘生长因子检测试剂盒（时间分辨荧光法）：由校准品、铕标示踪抗体、分析缓冲液、生物素抗体、链霉亲和素微孔板条、条形码标签等组成。用于定量测定孕妇血清中的胎盘生长因子。临床上用于辅助筛查妊娠妇女孕早期唐氏综合症风险。分类编码：6840。
（五）脂联素检测试剂盒（免疫比浊法）：由试剂1（缓冲液）和试剂2（乳胶抗体试剂）组成。用于体外定量检测人血清或血浆中脂联素浓度。临床上用于评估2型糖尿病和心血管疾病的风险。分类编码：6840。
（六）小而密低密度脂蛋白胆固醇测定试剂盒（直接清除法）：由试剂（含过氧化氢酶、胆固醇氧化酶、胆固醇脂酶、显色剂的缓冲液）和试剂（含过氧化物酶、酶稳定剂、显色剂的缓冲液）组成。用于体外定量检测人血清中小而密低密度脂蛋白胆固醇的含量，临床上用于动脉粥样硬化的辅助诊断。分类编码：6840。
（七）尿液游离LamBDa-轻链测定试剂盒（免疫比浊法）：由定标液、试剂缓冲液和试剂（游离Lambda-轻链抗血清）组成。用于体外定量测定尿液游离Lambda-轻链浓度，临床上用于单克隆丙种球蛋白增多症的辅助诊断。分类编码：6840。
三、作为第Ⅰ类医疗器械管理的产品（13个）
（一）制片染色一体机：通常由离心模块、制片模块、染色模块、控制系统等功能模块组成。用于病理分析前对人体细胞或细菌样本进行制片及染色。分类编码：6841。
（二）细胞分离制片染色一体机：通常由控制系统、过滤器、多路阀注射泵、贮液瓶、废液瓶等组成。适用于病理分析前对人体外周血或组织样本中细胞的分离、过滤、制片和染色。分类编码：6841。
（三）试剂卡孵育器：由塑料外壳、温控模块、电器元件组成。用于特定试剂卡的孵育。分类编码：6840。
（四）细胞过滤采集器：由软管、过滤收集病变细胞装置、抽吸装置组成。用于病理分析前对人体内脱落病变细胞的采集。分类编码：6841。
（五）液基细胞和微生物处理、保存试剂：主要由细胞保存液、细胞裂解液、提取液、稀释液、消化酶、防腐剂等及必要的细胞承载或制片器具组成。用于临床检验分析前细胞或微生物的保存、运输、提取、分离、沉淀、固定、制片等。分类编码：6840。
（六）病理分析前处理试剂：主要由组织固定液、组织脱水液、透明液、清洗剂等组成。用于病理分析前组织标本的固定、梯度脱水、透明、浸蜡和包埋制片。分类编码：6840。
（七）脱蜡液：由脱蜡液、防腐液、专用水等组成。用于对样本进行染色前预处理，去除石蜡包埋组织样本上的石蜡。分类编码：6840。
（八）触发缓冲液：由0.9％的氯化钠等渗溶液组成。与血小板功能分析仪及配套检测试剂盒配套使用，用于在检测前润湿生化活性膜。分类编码：6840。
（九）流式细胞分析用鞘液：由含盐缓冲液和防腐剂组成。用于与流式技术相关的分析中对样本进行检测时形成鞘流，以利于仪器进行计数分析。分类编码：6840。
（十）流式细胞分析用溶血剂：由浓度小于2％的甲醛和浓度小于15％的甘油组成。用于快速裂解人类外周血中红细胞，维持白细胞基本形态，临床上用于流式细胞仪检测前样本处理。分类编码：6840。
（十一）抗体稀释液：由缓冲液和防腐剂组成。用于临床检验时抗血清的稀释。分类编码：6840。
（十二）溶痰剂：由次氯酸钠、表面活性剂组成。用于抗酸染色前痰标本的均质化处理，以提高阳性检出率。分类编码：6840。
（十三）精液液化剂：由菠萝蛋白酶和蔗糖组成。用于精液检查前促进液化迟缓的精液标本的液化、降低精液的粘稠度。分类编码：6840。
四、不按照医疗器械管理的产品（1个）
瓷珠菌种保存管：由表面多孔的小瓷珠、胰蛋白脉大豆肉汤、甘油和蔗糖按一定比例混合组成。用于微生物检验中菌种的保存。
自本文件发布之日起，对于不作为医疗器械管理的，如已受理尚未完成注册审批的，食品药品监管部门应按规定不予注册，相关注册申请资料予以存档。尚在有效期内的医疗器械注册证书不得继续使用。
食品药品监管总局办公厅
2015年6月11日
今天发现，我在核酸技术区开辟的“DIG/Biotin标记的Southern/Northern杂交专题讨论”4个月，今天回帖数破100，点击达1500多次。能和大家一起分享和交流，并且得到共同提高，心情很是愉快。
所以想借PCR技术区的宝地，新开辟“环介导等温扩增技术(Loop-MediatedIsothermalAmplification,LAMP)专题讨论”。我做LAMP一年多了，有些经验。而最近，和我谈起LAMP的朋友明显增多。所以在这方面大家有什么心得、体会、资料和实验困难，希望能一起交流讨论。谢谢支持！
