Ordering
| Item | Catalog # | Description | Quantity | Price (USD) | ||
|---|---|---|---|---|---|---|
| Plasmid | 50467 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
| AAV2 | 50467-AAV2 | Virus (100 µL at titer ≥ 2×10¹² vg/mL)and Plasmid.More Information | Add to Cart | |||
| AAV8 | 50467-AAV8 | 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 sizew/o insert(bp)4818
- Total vector size (bp)8157
- Vector typeAAV
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)Stbl3
- Copy numberLow Copy
Gene/Insert
- Gene/Insert namehM4D(Gi)-IRES-mCitrine
- SpeciesH. sapiens (human)
- Insert Size (bp)2791
- MutationSee supplemental documents for DREADD mutations
- Entrez GeneCHRM4(a.k.a. HM4, M4R)
- PromoterCaMKIIa
- Tag/ Fusion Protein
- HA (N terminal on insert)
Cloning Information
- Cloning methodRestriction Enzyme
- 5′ cloning siteBamH I(not destroyed)
- 3′ cloning siteEcoR I(not destroyed)
- 5′ sequencing primerATGCTGACGAAGGCTCGCGA
- 3′ sequencing primerGCATTAAAGCAGCGTATCCACATAGC (Common Sequencing Primers)
Resource Information
- Supplemental Documents
- DREADD mutations
- Terms and Licenses
- UBMTA
- Ancillary Agreement for Plasmids Containing FP Materials
- Industry Terms
- Not Available to Industry
Depositor Comments
For more information, see: http://pdspit3.mml.unc.edu/projects/dreadd/wiki/WikiStart
Information for AAV2 (Catalog # 50467-AAV2)(Back to top)
Purpose
Ready-to-use AAV2 particles produced from pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine (#50467). In addition to the viral particles, you will also receive purified pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine plasmid DNA.
CaMKIIa-driven HA-tagged hM4D(Gi) and bicistronic mCitrine expression, for chemogenetic inhibition. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 2×10¹² 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, AAV2 cap gene
- BufferPBS + 0.001% Pluronic F-68 + 200 mM NaCl
- SerotypeAAV2
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCitrine
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 AAV8 (Catalog # 50467-AAV8)(Back to top)
Purpose
Ready-to-use AAV8 particles produced from pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine (#50467). In addition to the viral particles, you will also receive purified pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine plasmid DNA.
CaMKIIa-driven HA-tagged hM4D(Gi) and bicistronic mCitrine expression, for chemogenetic inhibition. 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, AAV8 cap gene
- BufferPBS + 0.001% Pluronic F-68 + 200 mM NaCl
- SerotypeAAV8
- PurificationIodixanol gradient ultracentrifugation
- Reporter GenemCitrine
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.
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[国家标准物质网]高频红外碳硫分析仪是一种常用的分析仪器，能快速、准确地测定钢、铁、合金、有色金属、水泥、矿石、玻璃及其它材料中碳、硫两元素的质量分数。高频红外碳硫分析仪在使用过程中需要进行定期维护，今天我们就来具体介绍一下高频红外碳硫分析仪的日常维护方法。
高频红外碳硫分析仪日常维护要点
1、高频红外碳硫分析仪分析样品所用的坩埚需要马弗炉中加热温度升到1000度到1200度，恒温2小时后，自然冷却至一定温度时，取出放干燥器中冷至室温备用。
2、分析低含量样品所用的钨粒需在恒温箱中，加热至200度烘2小时后，取出在干燥器中冷却后使用。
3、高频红外碳硫分析仪设置了高频输出功率调节，在未打开仪器电源之前，用万用表测试电网电压后，将换档开关拨至电网电压基本一致的档位上，开始调试仪器。方法是当然界电网电压为220V时，调至230V档为减小输出功率，调至210档为增加输出功率。
4、打开总氧气阀门，打开仪器电源，按技术员所教操作。
5、每天仪器示打开电源前或分析200个样品后，需清扫一次过滤网及整个炉腔。
6、每天应检查高频炉正前方的干燥剂和脱脂棉。一旦发现干燥剂1/3变红或结块，都应立即更换。脱脂棉长期使用1/3变黄后，也应立即更换。更换时将旋转试管下方的螺杆，使试管下降至能离开上端面为止，取出试管，更换后按原装好拧紧，检查不漏气为止。
7、每月应清洗过滤网一次，清洗方法是：按上述“清扫方法”取出过滤网后，再取出过滤网上、下两只密封圈，将粉尘清扫后，放入超声波清洗器中，在清洗槽内加入蒸馏水和洗洁精。打开超声波清洗器电源。30分钟后取出过滤网，用蒸馏水反复清洗后，用无水乙醇清洗。最后取出过滤网用电吹风干或放入烘箱烘干，装上、下O型密封圈，涂上真空硅脂，按原位老妈子，检查不漏气为止。
8、高频红外碳硫分析仪使用时每季度应检查经过压紧阀内部的硅橡胶管，若发现有老化或弹性不好时应立即更换。
摘自：国家标准物质网
protocol如下：
灌注取脑后，4%多聚甲醛4℃固定24-48h。修块3-4mm。
脱水（常温）：45%、55%、65%、75%、85%、95%、100%酒精各30min；
透明（常温）：（100%酒精+二甲苯）/210min，二甲苯①20min，二甲苯②至完全透明（约20min）
浸蜡（60℃）：（二甲苯+石蜡①）/210min，石蜡①30min，石蜡②40min；石蜡①为低熔点，石蜡②为高熔点
包埋：浸蜡后用石蜡②手动包埋；
切片厚度：6um、（8um也切的有）
展片：40℃水中展片；
烤片：60摄氏度烤片机侧烤约5min后吸去水分，转移至60℃温箱烤约4h；
脱蜡：烤片后趁蜡未凝固，进行脱蜡。二甲苯5min×2，100%酒精3min，95%酒精2min，
85%酒精2min，75%酒精2min，流水冲洗2min（放在缸子里用水泡的，换了几次水）；
染色：
HE染色：苏木精5-8min（新配为5min，时间长可为8min，用前过滤），流水冲洗3min
1%盐酸酒精分化5s，流水冲洗1min，0.1%伊红1-2min，
脱水：85%酒精2min，95%酒精2min，100%酒精2min，
100%无水乙醇：二甲苯（1:1）2min
透明：二甲苯（I）3min，二甲苯（II）3min；
CV染色：浸入CV染液中10-20min，
脱水：75%乙醇2min，85%乙醇2min，95%乙醇2min，100%无水乙醇2min，
100%无水乙醇：二甲苯（1:1）2min
透明：二甲苯（I）3min，二甲苯（II）3min；
封片：中性树胶封片（整个染色过程避免干片）。
部分HE染色、CV染色片子结果如图所示，基本上没有完整的片子（染色时，片子随着在液体里的时间脱片、烂片的程度加深），另CV染色着色浅。
请教战友是什么原因？哪里出了问题，该怎么解决？
主药:50mg
mcc:20mg
l-hpc:10mg
cms-na:10mg
5%pvp50%乙醇qs
外加
ms:0.5mg
cms-na:3mg
50度干燥
以上处方不粘冲,但因上处方的辅料与主药有相互作用,导致片子变黄
现换成以下处方
处方1
主药:50mg
预胶化淀粉:20mg
淀粉:20mg
8%淀粉浆:qs
外加
ms:0.5或1mg
滑石粉:3或5mg
60度干燥
以上处方粘冲,为何?
处方2
主药:50mg
预胶化淀粉:20mg
淀粉:10mg
甘露醇:10mg
8%淀粉浆:qs
外加
ms:0.5或1mg
滑石粉:3或5mg
60度干燥
以上处方粘冲,为何
谁用过亲水性改性的聚丙烯膜养过细胞，最好是whatman的，效果怎么样?十分感激！:)
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