上海沪震实业有限公司
扫一扫,手机逛起来
主营:ELISA试剂盒,人ELISA试剂盒,小鼠ELISA试剂盒,大鼠ELISA试剂盒,重组蛋白,金标试剂盒,**组化试剂盒,标准品,单克隆抗体,多克隆抗体,生化试剂,生物培养基,原代细胞,细胞株,实验室耗材,动物血清等产品
021-60345367
您现在的位置:
企业信息
9
  • 注册时间:2015-11-06
  • 联系人:游艳
  • 电话:021-60345367
  • 联系时,请说明仪表网看到的
  • Email:shhzsw01@163.com
在线询价
产品详情

pCL-Eco

  • 如果您对该产品感兴趣的话,可以
  • 产品名称:pCL-Eco
  • 产品型号:
  • 产品展商:HZbscience
  • 产品文档:无相关文档
  • 发布时间:2017-07-27
  • 在线询价
简单介绍
pCL-Eco的各批次质粒菌株发货前均经过严格的多重验证,如存在质量问题,请在收到产品的三个月内通知我司。收到pCL-Eco后请短暂离心,取2μl转化至对应感受态中,挑取单克隆重新提取质粒后使用。
产品描述

pCL-Eco载体基本信息

试剂名称: pCL-Eco
质粒类型: 哺乳动物载体;逆病毒包装;单质粒包装系统
高拷贝/低拷贝: 高拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV
载体大小: 12341 bp
5' 测序引物及序列: CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’
3' 测序引物及序列: --
载体标签:
载体抗性: Ampicillin
筛选标记:
克隆菌株: DH5α等
宿主细胞(系): 包装细胞系如293T
备注: 逆病毒包装载体pCL-Eco是单质粒包装系统,快速产生Helper-free、毒力高的逆病毒颗粒。
产品目录号: #12371
稳定性: 瞬表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pCL-Eco载体质粒图谱和多克隆位点信息

pCL-Eco

pCL-Eco载体简介

逆病毒包装载体pCL-Eco的使用方法 293T Growth Medium:  450 ml DMEM (JRH Biosciences)
   50 ml FBS (JRH Sciences)
   5 ml Glutamine (200 mM in saline, JRH Sciences)
   2.5 ml Gentamycin (10 mg/ml in water, Sigma) HC11 Growth Medium:  5 ml L-Glutamine (200 mM in saline, JRH Sciences)
  2.5 ml Bovine Insulin (1 mg/ml in 0.01 N HCL, Sigma)
  2.5 ml Gentamycin (10 mg/ml in water, Sigma)
  0.5 ml EGF (10 μg/ml in water, Gibco)
  50 ml FBS (JRH Sciences)
  440 ml RPMI (JRH Sciences) Day 0: Split 293T cells for transfection: Wash cells off plate with 10 ml Hanks and put into 15 ml Falcon tube
  Add 1 ml 10X trypsin and mix by inversion for about 1-2 minutes
  Spin at 1000 rpm in clinical centrifuge for 1-2 minutes
  Resuspend cells in 10 ml growth medium
  Count cells
  Plate cells at 1X106 cells per 100 mm dish Day 2: Transfect 293T cells: Plan transfection using 2 μg total DNA and 12 μl FuGene in 200 μl total 
  volume per 100 mm dish
  Dilute Fugene: 12 μl per reaction in serum free DMEM for total volume of
  200 μl

  Mix DNA plasmids in polypropelene tubes (Falcon #2063):  1 μg DNA of interest
     1 μg pCL-Eco Add 200 μl diluted FuGene dropwise to each DNA tube

  Incubate RT 15 min
  Feed cells with 5 ml fresh growth media and add FuGene/DNA mixture to media Day 3: Change medium: • Remove viral medium and CAREFULLY feed with 6 ml growth medium
  This step may reduce a cytostatic factor (produced after transfections) that
  can inhibit growth and infection of your target cells.

  Split HC11 cells for infection
  Seed HC11 cells at 300,000 cells per 100 mm dish
  If using coverslips, incubate with FBS for at least 1 hour prior to plating Day 4: Infect target (HC11) cells:  • Collect virus-containing media off 293T cells
  • Syringe filter media through .45 μm filter onto target cells
   (Evaporation O/N and loss in filter leaves about 5 ml per plate)
  • Add 10 μl 5mg/ml polybrene (final conc = 10 μg polybrene/ml media)
   (Store polybrene in aliquots to decrease freeze/thaw cycles)
  • Wrap plates in parafilm
  • Spin cells in clinical centrifuge 10 min at 1800 rpm
  • Rotate plate 1/3. Spin 10 min at 1800 rpm
  • Rotate plate 1/3. Spin 10 min at 1800 rpm
  • Remove viral media and replace with fresh growth media Day 6+: Harvest HC11 cells:  • Wash cells with HBSS
  • Scrape remaining cells, pellet and flash freeze for protein/RNA analysis

  Stain cells for beta-gal expression:

  Fixing Solution :
    1.35 ml 37% Formaldehyde
    0.2 ml Gluteraldehyde
    1X PBS to 25 mls

  Staining Solution:
    625μl 40 mg/ml X-gal in DMF
    50 μl 1M MgCL2
    750 μl 100 mM Potassium Ferricyanide
    750 μl 100 mM Potassium Ferrocyanide
    1X PBS to 25 mls
    [100 mM Potassium Ferrocyanide = 2.11 g/50ml PBS]
    [100 mM Potassium Ferricyanide = 1.64 g/50 ml PBS]

  Store at room temperature, protect from light.
    1. Make -gal solution and warm in 37°C water bath to prevent crystallization
    2. Aspirate off media
    3. Wash cells 1X PBS
    4. Fix cells 5 min at RT in fixing solution
    5. Wash 3X PBS
    6 Optional: Add PBS to cells and warm plate at 37°C 5 min (reduces
      crystallization)
    7. Stain cells with staining solution at 37°C for 1-24 hrs
    8. Store stained cells in 70% EtOH at 4°C
      (EMBO J. 5: pg 3133, 1986) Note:  HC11 and MEC primary culture cells are very adherent and can withstand
   the force of spinning the plates at 1800 rpm. We found that HC11 cells died at
   2200 rpm. However, this is cell type-dependent, and spinning speed should be
   optimized accordingly.
   Expect to see -gal staining at the periphery of the plate. During the spin
   infection the media is pushed to the outside. The cells in the center of the plate
   will not be exposed to virus and will not be efficiently infected with retrovirus. 

pCL-Eco载体序列

hz-4047R ACADM/MCAD  酰基辅酶A脱氢酶中链抗体
hz-5015R ACADL  酰基辅酶A脱氢酶长链抗体
hz-5018R ACADVL  酰基辅酶A脱氢酶很长链抗体
hz-5019R ACAT1  胆固醇酰基转移酶1抗体
hz-5020R SOAT2/ACAT2  胆固醇酰基转移酶2抗体
hz-2390R Aconitase 2/ACO2  铁调节蛋白2抗体
hz-5021R ACOX1  过氧化物酶酰基辅酶A氧化酶1抗体
hz-5030R ACOX2  过氧化物酶酰基辅酶A氧化酶2抗体
hz-3681R ADCY1  腺苷酸环化酶1抗体
hz-3920R ADCY2  腺苷酸环化酶2抗体
hz-4021R ADCY3  腺苷酸环化酶3抗体
hz-3921R ADCY4  腺苷酸环化酶4抗体
hz-3922R ADCY5  腺苷酸环化酶5抗体
hz-3923R ADCY6  腺苷酸环化酶6抗体
hz-3924R ADCY7  腺苷酸环化酶7抗体
hz-3925R ADCY8  腺苷酸环化酶8抗体
hz-3926R ADCY9  腺苷酸环化酶9抗体
hz-3916R ADCY10  腺苷酸环化酶10抗体
hz-0439R ACE1/CD143  血管紧张素转换酶ACE1抗体

关于我们| 易展动态| 易展荣誉| 易展服务| 易家文化| 英才集结号| 社会责任| 联系我们

备案号:粤ICP备11010883号| 公安机关备案号:44040202000312| 版权问题及信息删除: 0756-2183610  QQ: 服务QQ

Copyright?2004-2017  珠海市金信桥网络科技有限公司 版权所有

行业网站百强奖牌 搜索营销*有价值奖 中小企业电子商务**服务商
以上信息由企业自行提供,信息内容的真实性、准确性和合法性由相关企业负责,易展仪表展览网对此不承担任何保证责任。

沪公网安备 31011702004356号