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General information
Champion™ Competent Cells are chemically competent cells, which were prepared by SMOBIO to make E. coli perform excellent transformation efficiency. Standard transformation protocol is recommended for large plasmids or non-ampicillin selection. Time-saving transformation protocol is recommended for simple and rapid transformation. Champion™ Competent Cells are one of the fastest and simplest ready-to-use competent cell products in the world.
Kit contents
Champion™ Competent Cells
pUC19 Control Plasmid (5 μl, 10-4 μg/μl)
Champion™ Transformation Protocol Card
Shipping condition
Throughout the shipping process, the temperature is maintained under -70°C.
Storage and expiration
Champion™ Competent Cells must be stored between -70°C to -80°C. Subsequent freeze-thaw cycles will reduce transformation efficiency. If high efficiency is required for the experiment, do not use aliquots that have gone through several freeze-thaw cycles. The efficiency of Champion™ Competent Cells lasts for 1 year with proper storage.
Product Name | Genotype | Application |
Champion™ 109 High | e14(McrA-)recA1 endA1 gyrA96 hsdR17(rk-, mK+) phoA supE44 thi-1 relA1 ∆(lac-proAB) (F’traD36 proAB lacIqZ∆M15 | Appropriate for blue-white color and robotic screening. It is a fast growing strain forming visible colonies within 8~10 hours. |
Champion™ 21 | F’ ompT hsdSβ(rβ-mβ-) dcm gal λ (DE3) | Appropriate host for recombinant protein expression using T7-based expression vectors. |
Champion™ DH5α High | recA1 endA1 gyrA96 hsdR17(rk-, mK+) phoA supE44 relA1 thi-1 ∆(lacZYA-argF)U 169 φ80 ∆(lacZ)M15 F’ | Suitable for cloning with large plasmid and cDNA library construction, and also for blue-white colony selection. |
Items and ordering information
Product Name | Compatible to E. coli strain | Efficiency (cfu/μg) | Quantity | Cat. No. |
Champion™ 109 High | E. coli JM109 | >1 x 108 | 100 μl x 80 vials | CC0202 |
100 μl x 24 vials | CC0204 | |||
Champion™ 21 | E. coli BL21 (DE3) | >1 x 107 | 100 μl x 80 vials | CC2102 |
100 μl x 24 vials | CC2104 | |||
Champion™ DH5α High | E. coli DH5α | >3 x 108 | 100 μl x 80 vials | CC5202 |
100 μl x 24 vials | CC5204 |
Manual
Manual_Champion™ Competent Cell
SDS
SDS_Champion™ Competent Cell
Flyer
Champion™ Competent Cell
Protocol card
Will transformation efficiency be reduced when Champion™ competent cells are thawed, dispensed and refrozen repeatedly?
When Champion™ competent cells are thawed dispensed in aliquots and refrozen within 1 min, the transformation efficiency will be 10~20% reduced compared to the first time use.
What is the advantage of thawing competent cells with circulating water instead of still water?
Using circulating water can reduce the thawing time. Less thawing time shows higher efficiency than long thawing time.
Is there any difference of transformation efficiency between using plating beads, bending glass rod and plating loop?
The bending glass rod shows best efficiency and the plating loop show less efficiency than other method. For handle a lot samples at the same time the plating beads are recommended.
Is 1 second vortex critical for mixture the DNA?
One second vortex provides more reliable transformation efficiency (1.1 times compare with mixed by finger tapping)
Will heat shock affect the transformation efficiency?
Heat shock treatment will enhance the efficiency about 1~2X versus non-heat shock method.
Factors Affecting Transformation Efficiency
Thawing methods
Shorter thawing time is more efficient than a longer thawing time. Slow thawing caused by power shortages and unstable freezers will result in decreased efficiency.
Size of plasmid
Plasmid size affect the efficiency greatly. The efficiency of transforming a supercoiled 2.7 kb is approximately 100 times higher than that of a 10 Kb plasmid (using time-saving transformation protocol). For large plasmids (> 6 kb), standard transformation protocol is recommended.
Heat shock treatment
Heat shock treatment will enhance the efficiency about 1~2 folds versus non-heat shock method.
Plating methods
Bent glass rods show the greatest efficiency, while plating loop shows less efficiency than plating beads. When handling a large quantity of samples at the same time, plating beads are recommended.
Concentration of antibiotic
Antibiotic concentration is critical to use of the time-saving transformation protocol.
Antibiotic | Concentration |
Ampicillin (Ap) | 20 μg/ml |
Kanamycin (Km) | 25 μg/ml |
Tetracycline (Tc) | 7.5 μg/ml |
Chloramphenicol (Cm) | 20 μg/ml |
For plasmid size <6 Kb, the efficiency of kanamycin selection is usually 3~10 times less than the ampicillin selection. For plasmid size > 6 Kb, the efficiency of kanamycin selection is much lower than ampicillin. We suggest using the standard transformation protocol (with heat shock and recovery steps) to enhance the efficiency.
Product Name | Compatible to E. coli strain | Efficiency (cfu/μg) | Quantity | Cat. No. |
Champion™ 109 High | E. coli JM109 | >1 x 108 | 100 μl x 80 vials | CC0202 |
100 μl x 24 vials | CC0204 | |||
Champion™ 21 | E. coli BL21 (DE3) | >1 x 107 | 100 μl x 80 vials | CC2102 |
100 μl x 24 vials | CC2104 | |||
Champion™ DH5α High | E. coli DH5α | >3 x 108 | 100 μl x 80 vials | CC5202 |
100 μl x 24 vials | CC5204 |
High Fidelity PCR amplification
Amplification of target gene with HiFi™ DNA polymerase to minimize error rate.
[TF1000] SMO-HiFi™ DNA Polymerase, (1 U/μl, 100 U)
[TF3000] G-HiFi™ DNA Polymerase, (1 U/μl, 100 U)
Gel electrophoresis
Staining amplicons with safe fluorescent dyes, following by observation under blue-light illuminator to minimize damage of DNA amplicons and maximize successful cloning efficiency.
Safe fluorescent dyes
[NS1000] FluoroVue™ Nucleic Acid Gel Stain (10,000X), 500 μl
[DS1000] FluoroStain™ DNA Fluorescent Staining Dye (Green, 10,000X), 500 μl
[DL5000] FluoroDye™ DNA Fluorescent Loading Dye (Green, 6X), 1 ml
Blue-light illuminator
[VE0100] B-BOX™ Blue Light LED Epi-illuminator
Ligation
Blund-end PCR amplicons can directly ligate with PCR cloning vector.
[CV1000] GetClone™ PCR Cloning Vector, 20 Rxn
[CV1100] GetClone™ PCR Cloning Vector II, 20 Rxn
Colony PCR
Analyze colonies with PCR master mix to save preparation time.
[TP1100] ExcelTaq™ 5X PCR Master Mix, 200 Rxn
[TP1200] ExcelTaq™ 5X PCR Master Dye Mix, 200 Rxn
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QQ：1917490559空间：http://user.qzone.qq.com/1917490559
本实验室技术力量雄厚，具有先进的病理技术设备（全自动免疫组化染色机，全自动HE染色机、全自动脱水机、包埋机、石蜡切片机等）。主要的病理技术服务有：
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7、电镜技术（待定）。
（2）抗体孵育时间过长或温度较高：解决办法是，严格执行操作规程，最好随身佩带报时表或报时钟，及时提醒，避免因遗忘而造成时间延长。现在流行的二步法（Polymer）敏感性很高，要求一抗孵育的时间不是传统的1小时，而是30分钟，因此，要根据染色结果进行调整。
（3）DAB变质和显色时间太长：DAB最好现用现配，如有沉渣应进行过滤后再用。配制好的DAB不应存放时间太长，因为在没有酶的情况下，过氧化氢也会游离出氧原子与DAB产生反应而降低DAB的效力，未用完的DAB存放在冰箱里几天后再用这种似乎节约的办法是不可取的。DAB的显色最好在显微镜下监控，达到理想的染色程度时立即终止反应。不过当染色片太多时或用染色机时，这样做似乎不现实，但至少应对一些新的或少用的抗体显色时进行监控，避免显色时间过长。
（4）组织变干：修复液溢出后未及时补充液体、染色切片太多、动作太慢、忘记滴液、滴液流失等都是造成组织变干的原因。解决的办法是操作要认真仔细，采用DAKO笔或PAP Pen在组织周围画圈，可以有效的避免液体流失，也能提高操作速度。
（5）切片在缓冲液或修复液中浸泡时间太长（大于24小时）：原因上不清楚，但现象存在。有的实验室喜欢前一天将切片脱蜡至修复，第二天加抗体进行免疫组化染色，如果将装有切片和修复液的容器放在4ºC冰箱过夜，对结果无明显影响，如果放在室温，特别是炎热的夏天，会出现背景着色，因此，不可存放时间太长。
（6）一抗变质、质量差的多克隆抗体：注意抗体的有效期，过期的抗体要麽不显色要麽背景着色。用新买的抗体时最好设立阳性对照和用使用过的抗体作比较。
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2换用新鲜的二甲苯，再脱蜡5-10分钟。
3无水乙醇5分钟。
490%乙醇2分钟。
570%乙醇2分钟
6蒸馏水2分钟。
从烤箱出来需要冷却吗?
2、【仪器型号】：SX-500
3、【生产厂家】：日本TOMY
4、【检测适用范围】：高压蒸汽灭菌
5、【仪器使用优点、缺点】：必填项。其中缺点若觉得不便评论的大家可以不写，但若评价较为客观科学，我们还是鼓励的！我想仪器公司见到此帖也会根据我们的建议和评价来改进他们的工作！
6、【仪器外观描述或者图片】：见附件。
7、【说明书或者操作规范】：
对于传统的水平装入的高压蒸汽灭菌器，方形的提篮放入圆形的内腔，浪费了接近36%的内腔体积。Tomy产品的上部装入式设计充分地优化利用了内腔的空间，将要灭菌的物品直接放入内腔中，或将要灭菌的物品放入圆形提篮(标准附件)中再放入内腔中。顶部装入设计使样品的处理更加容易和样品处理量增大。顶部装入设计使高压蒸汽灭菌器可以安装在地面上，不需要支架和台面或节约了有限的台面。SX系列高压灭菌器顶部的操作仅需单手或单脚即可操作；打开顶盖只需用脚踩下位于底部的开关踏板，顶盖锁即打开，顶子即向上自动打开；关闭顶盖也非常轻松容易，只需将盖子压下即可。集成式设计，节约安装和使用空间。标准配备一个快速空冷系统，使高温高压后的温度能快速的降低到常温，极大地缩短了操作时间；同时该功能可用于较低温度的循环灭菌操作。可以采用定时钟设定定时启动，节约实验预备时间。操作面板上设置大型的指示灯通过颜色变化来指示设备状态。灭菌操作的选择和容易观察的实时状态显示。图形显示对灭菌过程的选择，有五种常用的操作模式供选择，并可通过召回方式设定。同时操作面板上的LCD闪烁显示整个灭菌过程的进行状态，图形和温度及时间显示，在灭菌操作完成后排气阀自动打开，速度可设置在6个级别内设定(对液体灭菌设为速度可变)
顶盖锁定功能，在灭菌操作过程中顶盖配备高水平的锁定装置，以避免可能事故发生。可变的温度设定，使设置100℃如同设置121℃一样容易，设置温度范围从45℃到135℃，分辨为1℃(加热模式可达104℃)。水位高度传感器安全装置的作用有效地防止了干加热的发生。灭菌过程中在温度和压力平衡时，排气的自动微调功能使腔内的条件达到最佳。
8、【仪器维护及保养知识】：无特殊。
9、【参考价格】：选填项。
10【使用后的评价】：容易操作，空间利用效率高。自动化程度高，安全高效。
