Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | ||
|---|---|---|---|---|---|---|
| Plasmid | 26968 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
| AAV1 | 26968-AAV1 | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV5 | 26968-AAV5 | Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV9 | 26968-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)
- Backbone manufacturerInvitrogen
- Backbone sizew/o insert(bp)5587
- Vector typeMammalian Expression, AAV
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)NEB Stable
- Growth instructionsStbl3 (rec A-) cells to avoid recombinations with the LTRs
- Copy numberHigh Copy
Gene/Insert
- Gene/Insert namehChR2(E123T/H134R)-EYFP
- Alt nameChETA-EYFP
- SpeciesH. sapiens (human)
- Insert Size (bp)1662
- MutationE123T, H134R
- Tag/ Fusion Protein
- EYFP (C terminal on insert)
Cloning Information
- Cloning methodRestriction Enzyme
- 5′ cloning siteAscI(not destroyed)
- 3′ cloning siteNheI(not destroyed)
- 5′ sequencing primerggccagcttggcacttgatg
- 3′ sequencing primerGCAATAGCATGATACAAAGG (Common Sequencing Primers)
Resource Information
- Terms and Licenses
- UBMTA
- Ancillary Agreement for Plasmids Containing FP Materials
- genOway Notice of RIghts
- Industry Terms
- Not Available to Industry
- Articles Citing this Plasmid
- 5 References
Depositor Comments
This plasmid contains the human elongation factor-1a promoter.
For additional information please visit - http://www.optogenetics.org
Information for AAV1 (Catalog # 26968-AAV1)(Back to top)
Purpose
Ready-to-use AAV1 particles produced from pAAV-Ef1a-DIO ChETA-EYFP (#26968). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO ChETA-EYFP plasmid DNA.
EF1a-driven, Cre-dependent, ChETA fused to EYFP for optogenetic 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 GeneEYFP (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 AAV5 (Catalog # 26968-AAV5)(Back to top)
Purpose
Ready-to-use AAV5 particles produced from pAAV-Ef1a-DIO ChETA-EYFP (#26968). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO ChETA-EYFP plasmid DNA.
EF1a-driven, Cre-dependent, ChETA fused to EYFP for optogenetic activation. 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
- Reporter GeneEYFP (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
- 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.
Data submitted about 26968-AAV5 by requesting scientist(s):
- Data 1: Rat, Brain parenchyma
Information for AAV9 (Catalog # 26968-AAV9)(Back to top)
Purpose
Ready-to-use AAV9 particles produced from pAAV-Ef1a-DIO ChETA-EYFP (#26968). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO ChETA-EYFP plasmid DNA.
EF1a-driven, Cre-dependent, ChETA fused to EYFP for optogenetic 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, AAV9 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV9
- PurificationIodixanol gradient ultracentrifugation
- Reporter GeneEYFP (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.
ebiomall.com
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TRIZOL的主要成分是苯酚。苯酚的主要作用是裂解细胞，使细胞中的蛋白，核酸物质解聚得到释放。苯酚虽可有效地变性蛋白质，但不能完全抑制RNA酶活性，因此TRIzol中还加入了8－羟基喹啉、异硫氰酸胍、β-巯基乙醇等来抑制内源和外源RNase（RNA酶）。
TRIZOL是从细胞和组织中提取总RNA的即用型试剂，在样品裂解或匀浆过程中，TRIZOL 能保持RNA完整性。加入氯仿后，溶液分为水相和有机相，RNA在水相中。取出水相，用异丙醇可沉淀回收RNA。
※0.1%的8－羟基喹啉可以抑制RNase，与氯仿联合使用可增强抑制作用。
※异硫氰酸胍属于解偶剂，是一类强力的蛋白质变性剂，可溶解蛋白质并使蛋白质二级结构消失，导致细胞结构降解，核蛋白迅速与核酸分离。
※β-巯基乙醇的主要作用是破坏RNase蛋白质中的二硫键。
TRIZOL是一种新型总RNA抽提试剂，可以直接从细胞或组织中提取总RNA。其含有苯酚、异硫氰酸胍等物质，能迅速破碎细胞并抑制细胞释放出的核酸酶。TRIZOL在破碎和溶解细胞时能保持RNA的完整性，因此对纯化RNA及标准化RNA的生产十分有用。
基本特点
Trizol试剂可以快速提取人、动物、植物、细菌不同组织的总RNA，该方法对少量的组织（50-100 mg）和细胞（5×106）以及大量的组织（≥1 g）和细胞（>107）均有较好的分离效果。TRIZOL试剂操作上的简单性允许同时处理多个的样品。所有的操作可以在一小时内完成。TRIZOL抽提的总RNA能够避免DNA和蛋白的污染。故而能够作RNA 印迹分析、斑点杂交、poly(A)+ 选择、体外翻译、RNA酶保护分析和分子克隆。并且利用DNA、RNA和蛋白质在不同溶液中的溶解性质，可以通过分层分别将不同层中的RNA（上层）、DNA（中层）、蛋白质（下层）分离纯化出来，效率极好。
Trizol试剂能促进不同种属不同分子量大小的多种RNA的析出。例如，从大鼠肝脏抽提的RNA琼脂糖凝胶电泳并用溴化乙啶染色，可见许多介于7 kb和15 kb之间不连续的高分子量条带，（mRNA和hnRNA成分）两条优势核糖体RNA条带位于~5 kb （28S）和~2 kb （18S），低分子量RNA介于0.1 和 0.3 kb之间 (tRNA,5S）。当抽提的RNA用TE稀释时其A260/A280比值≥1.8
TRIZOL
http://baike.baidu.com/view/533842.htm
步骤
A． 真核细胞和组织
自己需要的材料：
β－巯基乙醇、70％乙醇（DEPC水配置）、除RNase的枪头和EP管。
材料的最佳量
想得到最佳的产量和好的Hibind RNA柱的纯化效果，选用正确的细胞和组织量是最重要的。Hibind RNA柱的最大处理量是可变的，根据细胞和组织的类型。最大的结合能力是100ug的RNA。TRK裂解缓冲液的最大裂解能力是1*107个细胞或30mg的组织。
细胞的步骤
1． 用500ul的TRK裂解缓冲液裂解细胞（≤1*107），使细胞团松散，轻轻弹EP管或者用枪头吹旋，再用漩涡振荡混匀。
a) 使用前每1ml TRK裂解缓冲液加入20ul的β－巯基乙醇。
b) 如果是单层的组织培养细胞（成纤维细胞，内皮细胞等），可以直接在细胞培养器里裂解细胞。完全吸去培养基后直接加TRK裂解缓冲液至细胞中。加700ul的TRK到T35细颈瓶或250px的培养皿中，更小的培养器加500ul的TRK。将液体布满整个器皿表面，确保细胞裂解完全。将裂解产物转移至1.5mlEP管中。
c) 如果细胞是悬液培养，收集细胞（≤1,500rpm or 400*g）*5min，弃上清，加入TRK裂解液，漩涡振荡混匀，转移到1.5ml EP管中，进行步骤2。
2． 匀浆化裂解产物
“裂解和匀浆化样品”－第四页有详细的说明，如果细胞≤1*105，漩涡振荡1min。不完全使样品匀浆化会显著的减少RNA的产量还容易造成柱子堵塞。
a) 用匀浆器30 s，使样品匀浆化。
b) 用钝的针头的注射器（0.9mm直径），至少吹打5次是样品匀浆化。注射器要除RNase。
3． 将匀浆后的裂解产物转移至Homogenization Spin Column中，离心，≥1,4000*g, 3min,室温。将离心收集的流出液转移到新的EP管中。转到总RNA分离中第4步。
组织的步骤：
1． 用500ul的TRK裂解缓冲液裂解细胞（≤30mg），使用前每1ml TRK裂解缓冲液加入20ul的β－巯基乙醇。
500ulTRK裂解液最大裂解能力30mg，难破碎的组织大于20mg就用700ul的TRK裂解液。当组织量超出建议的最大量时，也不要超过40mg。
组织样品匀浆化参照第四页选择一种方法只用，除非是使用液氮，
2． 离心，≥1,5000*g, 5min, 室温。
3． 转移上清至，Homogenization Spin Column中，离心，≥1,3000*g, 3min,室温。将离心收集的流出液转移到新的EP管中。
总RNA分离：
4． 在裂解产物中加入50％体积（250ul－350ul）的无水乙醇（96％－100％）混匀，漩涡振荡20s。
5． 样品转移到Hibind RNA Mini column上，离心管的最大容量为750ul，加完乙醇后可能会形成沉淀。所以要充分振荡将所有的混合液加入到柱子上。室温离心，10,000*g,0.5-1min。弃去流出液（透过柱子流出到离心管里的液体）。
6． 将柱子放在一个新的2ml收集管中，加上300ul RNA wash buffer I。室温离心，10,000*g, 0.5-1min。同样弃掉流出液。
7． DNase I 消化（选择性）
在我的实验中没有做这一步。
8． 将柱子再放到一个新的2ml收集管中，加入400ul RNA wash buffer I, 室温离心，10,000*g, 30s，弃流出液。
9． 将柱子放到2ml收集管中，加入500ul RNA wash buffer II（乙醇稀释过的），室温离心，10,000*g, 30s，弃流出液。注意：使用前RNA wash buffer II 必须要用无水乙醇稀释(48ml)，按照标签上的说明。
10． 用500ul RNA wash buffer II洗涤一次柱子，同上一步骤一样。室温离心，10,000*g, 30s，弃流出液。然后在空收集管中，以最大转速离心柱子，≥12,000*g, 2min, 室温，使的HiBind 基质完全干燥。
11． 转移柱子到一个新的1.5ml的Ep管中，（试剂盒中没有提供），加入50－100ul的DEPC水洗脱柱子（试剂盒中提供）。确保水直接加到了柱子的基质上。10,000*, 1min, 室温。如果RNA的总量大于30ug，可以进行二次洗脱。
注意：可以选择性地，用大一点的体积洗脱RNA。如果进行二次洗脱，总RNA的量会增加，但是浓度会降低，因为80％以上的RNA会在第一次时被回收。在加柱子前将水加热到65℃并室温孵育柱子5min会增加产量。
这里有关于miRNA专题报道，可以看看
不懂得请别粘贴那些有的没的，浪费资源
采用的给全部分
－－本来，看好丹麦Exiqon公司，Qiagen的两个试剂盒，无奈经费有限，这两个盒子很不错，价格也不菲，有米的兄弟姐妹可以买。
－－另外查询到国产上海诺伦公司的血液(血清/血浆)总RNA抽提试剂盒－－广州这边用的人不多，不知道上海那边的兄弟姐妹有什么经验？
－－－还有，北京康为世纪公司的游离RNA（血清血浆尿液）提取试剂，货号：CW2281，也比较便宜，但是俺心中也没有底，请大家给点意见。
－－－已经订购了LIFETECHNOLOGY的TrizolLS专门提取体液RNA的试剂（100毫升2000元人民币，也不菲），准备预试验看看提取RNA的质量。(Lifetechnology公司的AM1556也不错，但是也不便宜，每个盒子40次/2590元）
－－－－但是还是希望有试剂盒，比较简单，快捷，最重要的是样本量太大，要分离400份左右的血清。
