![SMOBIO/[CK1000] Champion™ E. coli Transformation Kit, 200 Rxn/s)"}]]>
</ul>
<a class=b](images/SMOBIO/image.jpg)
Description
Champion™ E. coli Transformation Kit provides an easy method for rapid preparation of chemically competent cells with high transformation efficiency from fresh culture, overnight culture, or even directly from bacterial colonies on the plate. The competent cell preparation method eliminates the requirement of time-wasting wash step. In addition, preparation of competent cells from overnight culture or directly from bacterial colonies provides flexibility to cloning experiment. The resultant competent cells can be immediately used or stored at -70°C for one year. This kit includes a specialized SMO-Broth™ medium and a unique Champion™ CC Buffer for culturing and preparing competent cells efficiently. Following the simple and quick competent cell preparation protocol from fresh culture, the transformation efficiency is typically ranged from 108–109 cfu/μg transformants/μg of pUC19 plasmid DNA, but varies depending on the E. coli strains. The resultant competent cells can be further transformed using time-saving transformation protocol, eliminating the requirement of heat-shock and recovery steps.
Features
Flexible– fresh culture, overnight culture, 4°C stored liquid culture or even colonies on agar plate can be used for transformation.
Fast and Easy– only few steps for preparation; suitable for time-saving transformation
High efficiency– up to 109 cfu/μg
Personalization– suitable for most E. coli strains
Kit Contents
Component | Volume | |||||
Champion™ CC Buffer | 20 ml | |||||
SMO-Broth™ | 2 x 100 ml | |||||
pUC19 Control Plasmid (10-4 μg/μl) | 5 µl | |||||
Instruction Manual | 1 | |||||
Champion™ Competent CellPreparation Card | 1
|
Storage
4°C for 12 months
Prepare Competent Cells with High Transformation Efficiency Using Champion™ E.coli Transformation Kit
Prepare Competent Cells Directly from E.coli Colonies Using Champion™ E.coli Transformation Kit
Time-saving Transformation Protocol
Kit Contents
Component | Volume |
Champion™ CC Buffer | 20 ml |
SMO-Broth™ | 2 x 100 ml |
pUC19 Control Plasmid (10-4 μg/μl) | 5 µl |
Instruction Manual | 1 |
Champion™ Competent CellPreparation Card | 1 |
Storage 4°C for 12 months |
Manual
Manual_CK1000_Champion™ E. coli Transformation Kit
SDS
SDS_CK1000
Protocol card
Can Champion™ E. coli Transformation Kit be used to make competent cell of other bacteria?
Champion™ E. coli Transformation Kit are specially designed for making E.coli competent cells. Therefore, Champion™ E. coli Transformation Kit is not recommend to used for make other bacterial competent cells.
What E.coli. strain is compatible to Champion™ E. coli Transformation Kit?
Most E.coli strains are compatible to Champion™ E. coli Transformation Kit. However, different E. coli strains vary in their ability to be transformed with DNA.
Efficiency of strains and their derivatives is listed below, when prepared with the Champion™ E. coli Transformation Kit.
E. coli strains | Efficiency (cfu/μg) |
JM109 | ~5x108 |
XL-1 blue | ~3x108 |
DH5α | ~5x108 |
stbl 3 | ~2x108 |
BL21 | ~1x107 |
Rosetta 2 | ~1x107 |
*E. coli strains are liquid cultured following standard protocol for preparation of competent cells (at 25°C until OD600 reached ~0.5).
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.
Is it necessary to change the transformation procedure for transforming a large plasmid?
For large plasmid (> 6 kb), the heat shock method will significantly improves the efficiency and the recovery procedure also significantly improves the efficiency.
How to reduce the interference of the satellite colonies?
Using proper antibiotics of suitable concentration or using fresh antibiotics.
Tips for Making High Competence of E. coli
Culture Condition
E. coli cells present high competence when freshly cultured from 1/100~1/20 dilution of overnight culture and incubated at 16~25°C prior to preparation. Higher temperature (37°C) leads to competence decrease.
Temperature | OD600 | Culturetime | Efficiency(cfu/μg) |
16°C | 0.4~0.8 | 12~20hr | ~1x109 |
25°C | 0.4~0.8 | 3~8hr | ~5x108 |
37°C | 0.4~0.8 | 1~1.5hr | ~1x108 |
25°C | 1~2.5 | 4~10hr | ~3x108 |
E. coli cells in liquid culture are higher competent than colonies on plate.
Culture Condition | Efficiency (cfu/μg) |
Liquidculture (fresh or overnight culture) | 108~109 |
Freshcolonies (grown overnight at 37°C) | ~2x106 |
Oldcolonies (4°C storedfor 7 days) | ~5x105 |
E. coli Strains
Different E. coli strains vary in their ability to be transformed with DNA. Efficiency of strains and their derivatives is listed below, when prepared with the Champion™ E. coli Transformation Kit.
E. coli strains | Efficiency (cfu/μg) |
JM109 | ~5x108 |
XL-1 blue | ~3x108 |
DH5α | ~5x108 |
stbl 3 | ~2x108 |
BL21 | ~1x107 |
Rosetta 2 | ~1x107 |
*E. coli strains are liquid cultured following standard protocol for preparation of competent cells (at 25°C until OD600 reached ~0.5).
Storage Condition
To maintain high competence, E. coli should be stored at -70°C. Higher temperature (above -50°C) leads to competence decrease.
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.
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
ebiomall.com
>
>
>
>
>
>
>
>
>
（第1张图和第2张图是经旋转后方向恰好相反的两张图，上矢状窦等静脉也显示了）
女患，70岁，大专毕业，我院退休药师。7-8年前患脑梗（具体不详）。记忆力差5-6年，近记忆为著，几次做饭后忘记关火，将炉具的台板（玻璃的）烧裂。近5-6年来，病人每年住院彻底检查治疗1次（主要是使用活血化瘀及营养脑细胞药）。平素，病人双耳听力略差，睡眠欠佳、便秘、尿频，偶从卧位坐起时视蒙。既往有时血压略高，未降压治疗。本次为“通血管”再来住院。查体：血压：140/80mmHg,双耳听力略差，近记忆力差，但智力（MMSE：26分)正常，颅神经未见异常，四肢肌力、感觉未见异常，植物神经未查，双掌颌反射（+），双下肢病理反射均阴性。血常规：淋巴细胞比率略高，余均正常；尿常规、心电图、癌胚抗原均正常；凝血四项：除凝血酶时间略长外均正常；生化全项：除总胆固醇略高外均正常；超声心动图：老年瓣退行性改变，左室舒张功能减退，房膜瘤可能。彩超：双侧颈动脉粥样硬化形成。头MRA:右大脑中动脉及其分支未显示。
问题：1MRI怎么会这样逍遥？病人无肢体瘫，生活自理如常人。
2为明确头MRA:右大脑中动脉及其分支未显示的病因和程度，除做DSA外，还需做什么？
3如不做DSA，给他订类药、阿斯匹林及活血化瘀的中药可不可以？
教。
版主laocao留言:
患者的年龄，病史，症状，辅助检查传上来，便于进一步讨论，谢谢！
