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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.
Genotypes and applications
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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它一般在医院、制药企业使用。
试剂盒使用示例： 试剂盒的产生正是为了使实验人员能够摆脱繁重的试剂配制及优化过程，所以试剂盒中一般配备有相应的使用说明书
主要有： 1.磁珠法核酸提取试剂盒2.磁珠法提取DNA试剂盒3.DNA提取试剂盒（离心柱法）4.磁珠法RNA提取试剂盒5.RNA提取试剂盒（离心柱法）6.磁珠法植物基因组DNA提取试剂盒7.反转录试剂盒8.胶回收试剂盒9.PCR纯化试剂盒10.病毒核酸提取试剂盒
如果PCR产物不是很纯，或者PCR扩增条带比较小，PCR产物前面又有较多引物二聚体时，用胶回收，其余用PCR产物纯化试剂盒。
pcr纯化试剂盒和胶回收试剂盒的区别：
PCR纯化试剂盒：是直接水溶解的PAC产物就可以回收，回收效率高，但是只适合单一条带需要纯化测序的时候使用。
PCR凝胶试剂盒：是在PCR产物是混合物，有多条杂带的情况下，先跑胶将杂带分离，然后在将所要的条带位置的胶切下回收，后者的回收效率低，但是很纯净。
胶回收试剂盒操作步骤：
配制琼脂糖EB凝胶,电泳以分离DNA片段。任何类型或等级的琼脂糖都可以使用。
电泳足够时间后，在紫外灯下小心地把所需的DNA的片段切下来。并尽量去除多余的凝胶。
称取空离心管的重量，切下带目的片段的凝胶装在1.5ml离心管中并称其重量，求出凝胶块的重量，近似地确定其体积。一般情况下，凝胶的密度为1g/ml，于是凝胶的体积与重量的关系可按下面换算：凝胶薄片的重量为0.2g 则其体积为0.2ml；加入等倍凝胶体积的Binding Buffer，把混合物置于55℃~65℃水浴中温浴7min至凝胶完全融化，其间每隔2-3分钟混匀一次；
转移700μl的DNA-琼脂糖溶液到一个HiBindTM DNA柱子，并把柱子装在一个干净的2ml收集管内，室温下，10,000×g离心1min，弃去液体。
将柱子重新套回收集管中，加300μl Binding Buffer至HiBind DNA 柱子中；室温下，10,000×g离心 1分钟，去弃滤出液；这一步相当关键，不要忽略此步。
将柱子重新套回收集管中，加入700μl SPW Wash buffer至HiBind DNA柱子中，室温下，10,000×g离心1分钟，去弃滤出液；注：SPW Wash buffer在使用前必须按瓶子标鉴要求用无水乙醇进行稀释。
将柱子重新套回收集管中，重复加入700μl SPW Wash buffer至HiBind DNA柱子中，室温下，10,000×g离心1分钟，去弃滤出液；
弃去液体，将空柱子重新套回收集管中，10,000×g离心1min以甩干柱基质残余的液体。
这步可以去除柱子基质上残余的乙醇，不要省略此步―――对得到好的DNA产量是十分重要的。
把柱子装在一个干净的1.5ml离心管上，加入30~50μl洗脱液或灭菌水上柱子膜上，10,000×g离心1分钟，离心管中的溶液就是纯化的DNA产物，保存于-20度。
病毒液通过pre-filterdisc的时候是不是要制造一个真空环境产生压力差使液体通过？这个时候是用自备的针管还是试剂盒配的5毫升的针管啊？
