Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | ||
|---|---|---|---|---|---|---|
| Plasmid | 68720 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
| AAV1 | 68720-AAV1 | 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)
- Total vector size (bp)6958
- Vector typeAAV
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)Stbl3
- Growth instructionsDue to ease of recombination, AAV and lentivirus vectors should be amplified in a recombination deficient bacteria strain such as Invitrogen's OneShot Stbl3 cells. Check for integrety of ITR sites with SmaI digest.
- Copy numberLow Copy
Gene/Insert
- Gene/Insert namemRuby2-P2A-GCaMP6s
- SpeciesR. norvegicus (rat), G. gallus (chicken); A. victoria (jellyfish)
- Insert Size (bp)2124
- MutationCre-dependent expression from inverted open reading frame (i.e. 'FLEXED')
- PromoterhSyn1-FLEX
Cloning Information
- Cloning methodUnknown
- 5′ sequencing primeractcagcgctgcctcagtct
- 3′ sequencing primergtttgtacaaatgatgacagcgaag (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
- Article Citing this Plasmid
- 1 Reference
Depositor Comments
mRuby2: Article: Improving FRET dynamic range with bright green and red fluorescent proteins. Lam et al (Nat Methods. 2012 Sep 9. doi: 10.1038/nmeth.2171. PubMed) Addgene Plasmid 40260
GCaMP6s: Article: Ultrasensitive fluorescent proteins for imaging neuronal activity. Chen et al (Nature. 2013 Jul 18;499(7458):295-300. doi: 10.1038/nature12354. PubMed)Addgene Plasmid 40753
Information for AAV1 (Catalog # 68720-AAV1)(Back to top)
Purpose
Ready-to-use AAV1 particles produced from pAAV-hSyn1-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (#68720). In addition to the viral particles, you will also receive purified pAAV-hSyn1-Flex-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA plasmid DNA.
Cre-dependent GCaMP6s calcium sensor and bicistronic, physically separate mRuby2 expression under a human synapsin1 promoter. 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 GenemRuby2 (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.
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大家做溶出曲线稀释溶液时有某有再用移液枪？移液枪量取1ml的体积准确不？
像乙醇、二氯甲烷、乙酸乙酯、丙酮这类毒性较低，挥发性不是很大的，移液枪取肯定没问题，但是一个移液枪最多也就取10ml，我觉用这么精准的仪器来取溶剂，既麻烦又没必要；
像一些吡啶、哌啶类的毒性较大的，你用移液枪取后很容易有残留，造成污染就不好了；
还有就是性质不稳定的例如三氯氧磷啊、二氯亚砜啊这类，挥发枪，和空气中水反应。。。最好不要用移液器，你想取样精密点，就查密度称重取就好了。。。
还有很多例如氯苯之类的密度大于水的有机溶剂，粘度又小，用移液枪取很容易滴漏，造成损失误差；
你说一句“有机溶剂”，但其实情况很复杂的。。。所以，具体情况具体分析
我们每天长时间都在实验室里和移液器（俗称“枪”）打交道，枪已经成为实验室标配，因为它是很多实验过程和分析的基础。但正因为如此，我们都认为它很简单，就像走路和呼吸一样自然，不需要你花任何心思，它就可以做你想做的事情。但事实真的是这样吗？
事实上，枪虽小但往往关系到实验结果的准确性，有小伙伴经常抱怨枪老出问题：
1.用了一段时间就不准漏液了？
2.液体容易倒吸？
3.移液排不干净？……
到底是哪里出问题了呢，你有没有碰到这样的迷之困惑？
当当当，福利到，来自梅特勒-托利多瑞宁移液技术专家自告奋勇来解答大伙儿的困惑，有任何关于移液器使用的问题欢迎来和我们探讨共同进步哟~
最后感谢丁香园支持本活动！
我们会每天抽时间统一回复大家的问题，专家答疑截止12月31日哦！
2、吸液：用右手的拇指和中指捏住移液管的上端，将管的下口插入欲吸取的溶液中，插入
不要太浅或太深，一般为10～20mm处，太浅会产生吸空，把溶液吸到洗耳球内弄脏溶液，
太深又会在管外沾附溶液过多。左手拿洗耳球，接在管的上口把溶液慢慢吸入，先吸入该管 容量的1/3
左右，用右手的食指按住管口，取出，横持，并转动管子使溶液接触到刻度以上 部位，以置换内壁的水分，然后将溶液从管的下口放出并弃去，如此用反复洗3
次后，即可 吸取溶液至刻度以上，立即用右手的食指按住管口。 3、调节液面：将移液管向上提升离开液面，管的末端仍靠在盛溶液器皿的内壁上，管身保
持直立，略为放松食指（有时可微微转动吸管）使管内溶液慢慢从下口流出，直至溶液的弯
月面底部与标线相切为止，立即用食指压紧管口。将尖端的液滴靠壁去掉，移出移液管，插 入承接溶液的器皿中。
4、放出溶液：承接溶液的器皿如是锥形瓶，应使锥形瓶倾斜30°，移液管直立，管下端紧
靠锥形瓶内壁，稍松开食指，让溶液沿瓶壁慢慢流下，流完后管尖端接触瓶内壁约15 秒后，
再将移液管移去，残留在管末端的少量溶液，不可用外力强使其流出，因较准时已考虑了末 端保留的溶液的体积。
一．设定移液体积：1.从大体积调节到小体积时，为正常调节方法，逆时针旋转刻度即可；2.从小体积调节至大体积时，可先顺时针调至超过设定体积的刻度，再回调至设定体积，这样可以保证最佳的精确度。
二．装配移液器吸头：1.单道移液器，将移液端垂直插入吸头，左右微微转动，上紧即可2.用移液器反复撞击吸头来上紧的方法是不可取的，这样操作会导致移液器部件因强烈撞击而松散，严重的情况会导致调节刻度的旋钮卡住。3.多道移液器，将移液器的第一道对准第一个吸头，倾斜插入，前后稍许摇动上紧，吸头插入后略超过O型环即可。
从上理解，
移液枪增量是增加移液体积。
