Highlights
- Simple 20 Second Transformation: No heat shock! Just add DNA and spread on plate.
- High Transformation Efficiencies: Achieve 108 - 109 per µg of plasmid DNA.
- Versatile: Excellent for general cloning, blue-white screening, and plasmid isolation.
Description
Additional Info | Partly restriction-deficient; good strain for cloning repetitive DNA (recA-). Suppresses many amber mutations when glutamine is available but not the S100 or S7 mutation of λ, e.g.,λgt11. Can be used for M13 cloning/sequencing and blue/white screening. |
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Genotype | F`[traD36 proA+B+ laclq Δ(lacZ)M15] Δ(lac-proAB) glnV44 (supE44) e14- (McrA-) thi gyrA96 (NalR) endA1 hsdR17(rk- mk+) relA1 recA1 |
Processing Time | 20 Seconds |
Product Storage | -70°C to -80°C |
Transformation Efficiency | 108 - 109 transformants per µg of plasmid DNA |
Q1: Are competent cells GMOs?
All our competent cells are classified into Biosafety level 1 and are not genetic modified organisms. Only when transformed with a plasmid they become GMOs.
Q2: Are the Mix & Go! strains dam+ and dcm+?
Most cloning strains will be dam+/dcm+ unless specifically noted in the genotype.
Q3: Do the Mix & Go! strains methylate DNA?
Yes
Q4: Which strains are equivalent to the Zymo strains?
DH5α is equivalent to Zymo 5α. DH10B, Top10, and One Shot Top10 are equivalent to Zymo 10B.For XL-21 Blue, JM109 is the closest match and for Stbl3, HB101 is the closest match.
Q5: How to reduce satellite colonies on agar plates?
– Prepare fresh agar plates– Use more antibiotics in plates– Incubate plates for a shorter time after plating cells
Q6: Is it possible to dilute the competent cells?
We do not recommend diluting the competent cells. We recommend using less DNA to transform cells, or aliquot cells in smaller volumes before transformation. If absolutely necessary, cold 1X Competent Buffer (Mix & Go Transformation Kit, T3001 & T3002) should be used in the dilution.
Q7: Which antibiotics can be used with the Mix & Go! procedure?
No outgrowth is necessary when using Ampicillin or Carbenicillin for selection. However, an outgrowth step is required when using Chloramphenicol, Kanamycin, and Tetracycline because of the mode of action of the antibiotic itself. We recommend the following procedure for the outgrowth step:1. Incubate cells on ice for 5-10 min after addition of plasmid 2. Add 4 volumes of SOC media3. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm4. Spread on a pre-warmed culture plate containing the appropriate antibiotic
Q8: Which Plasmid Size can be used for transformation?
For Zymo 5α and Zymo 10B up to 20kb. However, transformation efficiency decreases proportionally from 10-20kb. Above 20kb, cells are difficult to transform. JM109, HB101, XJa, XJa (DE3), XJb, XJb (DE3) and TG1 can handle constructs up to 10kb.
Q9: Which is the recommended DNA concentration and volume for transformation?
There really is no maximum or minimum recommended DNA concentration, but we use 10 pg for quality control. However, the volume of DNA added should not exceed 5% of the cells total volume; the efficiency can decrease several fold as the volume of DNA used increases. If the DNA sample is too diluted, use our DNA Clean & Concentrator.
Q10: What are some tips to improve transformation efficiency?
1. Thaw cells on ice, not room temperature.2. Incubate cells and DNA mixture on ice, not at room temperature.However, do not incubate longer then 1 hour.3. Ensure cells are still frozen when received.4. Pre-warm the culture plates at 37°C for at least 30 minutes.5. Prepare fresh LB agar plates containing the appropriate antibiotic.6. Prepare a new DNA sample.7. Store the cells at -80°C (not 4°C or -20°C). If the freezer breaks, the cells should be OK as long as the temp does not go higher than -50°C.8. Avoid freeze/thaw cycles.
Q11: How will a heat-shock affect my Transformation Efficiency?
Heat shock is not necessary, however sometimes it can be beneficiary when preparing libraries or transforming XJb Autolysis E. coli strains.We recommend the following protocol for Heat Shock with Outgrowth: 1. Incubate cells on ice for 5-10 min after addition of plasmid. 2. Incubate cells at 42°C for 45 seconds.3. Add 450 ml of SOC to the cells. 4. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm.5. Spread on a pre-warmed culture plate containing the appropriate antibiotic.
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缺点是人工计数可能发生的随机误差较大。
免疫组化、免疫荧光、Westernblotting、RT-PCR和细胞计数方法已经渗透到大多数研究生试验课题中,这些形态学试验方法的结果如何定量一直困扰着许多研究生,而这些图像的准确数字化分析结果远超过单一图片所显示结果的说服力,尤其在发高影响因子文章时。为解决图像分析过程中的问题困扰,我版特邀请目前在蚂蚁淘论坛很有名气的hbchendl老师给大家做一讲座和解惑,而本帖主要用于征集”图像分析疑难问题”和活动建议。
一、专家介绍——hbchendl战友提供
这次接受形态学版主邀请来作关于图像分析的讲座,内心里多少有点惶恐,看以前在这里作讲座的都是各个领域的专家学者,而我却只是一个实验技术人员。当然是属于有点实践经验的实验技术人员。
本人从事显微镜的操作维护管理有很多年,从2000年开始接触到图像分析软件Image-proplus4.5,开始只是作一些简单的图像分析测量,真正深入理解IPP软件还是在进入蚂蚁淘之后,确实在蚂蚁淘里开拓了眼界。然后在工作中做了许多图象分析的工作之后,对软件使用比较熟悉了。正好当时需要培训其他同事学习使用IPP,于是就有了《手把手教你使用Image-proplus》这一组帖子,当然也是为了挣点积分能看更多的好帖子。这组帖子有些影响,甚至把MediaCybernetics公司的人给招来了,他们又教了我一些使用IPP的技术与方法,还为分析免疫组化图片制作了一个宏操作程序。由我进行程序分析的调试,最后就有了那个给大家带来巨大方便的pathology.ipm宏操作程序。
另一方面,在蚂蚁淘的这些年,通过参与其他战友的分析测量工作也使我得到更多的机会来进一步理解图像分析。也发现了那一组《手把手教你使用Image-pro》中还有一些错误,曾经作过一些修改,但现在基本上没法改了。所以最近又在蚂蚁淘博客上开博客来写《手把手教你使用Image-proplus》第二版,还没写完。平时经常有同学询问关于图像分析的事。但是一个最常见的现象是提问时说得太简单,我无法从中知道对方想测量什么,当然也就无法讨论起来。这次希望大家在讨论时先介绍一下自己所要测量的内容,并且把图片帖上,这样我可以帮助大家分析一下,更好地解答问题。
最后感谢大家的关注,希望大家通过这次活动能各得其益。特别是感谢形态学版主wh2008的组织协调工作。
hbchendl战友的图像分析相关精华链接汇总:第二版手把手教你使用Image-proplus第二版手把手教你使用教程_pdf版本下载链接(感谢doctor_li战友和hbchendl战友的贡献)
二、活动安排
1、本帖主要用于征集IPP图像分析相关难题和活动建议——1~2周后由版主或热心战友归类汇总:
2、由hbchendl战友新建帖子,讲述IPP图像分析原理、操作要点和收集的难题解答以及现场答疑活动等,时间1~2周;
3、wh2008版主对此次活动总结,并决定是否继续开展以下候选讲座。
三、参与战友有奖
对于积极参与此次活动的热心战友并有以下工作成绩之一的战友将给予积分或投票奖励:参与共享图像分析相关的珍贵资源、提出问题被征集、正确解答战友问题、提出很好建议并被采纳、积极参与组织活动并取得优异成绩者等。
四、候选讲座:
免疫组化试验技术(拟由wh2008版主支持)、病理常规阅片技巧、常规染色方法交流等等
请大家多多提意见。
最新形态版系列有奖活动:
理论篇:成语形态新解成语故事形态新编应助有奖推荐晋级
图片篇:精美解剖学图片展
文献篇:文献阅读活动规则
实验篇:2009形态版第一场实验技术讲座(IPP图像分析原理及其问题答疑)即将开始——“图像分析疑难问题”征集活动正在进行中[精华]【原创】第三讲:电泳图片光密度分析原理与方法
需要做脂肪代谢实验,打算通过在心肌新鲜组织匀浆中加入核素标记的脂肪酸代谢底物([14C]-Palmitate),通过液体闪烁计数仪测定氧化产物[14C]CO2的放射性计数来反应脂肪酸的氧化代谢率。但是实验室只有γ放射免疫计数仪,请问能否用其测量“软β射线”14C;仪器需要作何调整吗???
1、同样的测量条件下,如测量相对几何位置相同,放射源相同,放射性测量计数率扣除本底后与活度在统计误差内成正比;
2、同样活度不同核素放射源,在其它条件相同的情况下放射性测量计数率不一定相同。因为不同核素衰变各能量分支比不同、粒子不同、能量不同,有的衰变子体也有放射性。
3、放射性测量计数率与计数器的探测效率相关。简单认为:活度=(计数率-本底计数率)/效率。其中效率的标定要与测量条件一致。
因为声的多普勒效应,用专用仪器在空气里测频率似乎变得不可靠。 最简单直接的方法,用频率特性仪,接换能器正负极测量读数 或者拿一片超声波振子接受对方发射的超声波进行放大后,输入到整形电路,再输入到计数器,就可以测量频率了
能不能用kappa检验,我看了论坛上的帖子,好像是不行的。但是看到一篇文章,计数资料却用了kappa检验,请问能否这样使用,如果能的话,应该用什么软件,怎么计算。谢谢
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