g***j
发帖数: 40861 | 1 做转化的时候我发现有时候感受态细胞即使没转入带kanamycin的质粒也可以在
kanamycin的平板上生长。
什么道理？ |
|
q*******8
发帖数: 768 | 2 平板不新鲜了，抗生素过期，浓度算错，感受态本来就抗kanamycin。。。 |
|
u***************r
发帖数: 11227 | 3 学医的同学们给解释一下，难道这种悲剧是不可避免的吗？有没有象测试青霉素过敏的
办法鉴别对耳毒性药物的敏感性？有没有安全的替代药物？
【 以下文字转载自 Medicine 讨论区 】
【 原文由 forestwolf 所发表 】
I have a friend whose nephew had hearing
disability by misusage of Kanamycin. Her nephew's hearing disability was
gotten several months after he was born. Now the child is about three
years old. At that time, the child got a fever and the doctor had him take
the Kanamycin,maybe overdose. Gradually,the child was found to hardly hear
any sound. It is a grievous pain for the parents to |
|
i********n
发帖数: 171 | 4 send your CV and manuscript # to [email protected]/* */
1. In vitro anti-oxidative activity and anti-fibrosis potentials of
compounds derived from traditional herbal medicines
2. Jesridonin in Combination with Paclitaxel Demonstrates Synergistic
Anti-tumor Activity in Human Esophageal Carcinoma Cells
3. Abnormal expression of fork-head box P3 suppresses migration and
invasion and is associated with the prognosis of cholangiocarcinoma
4. Polyethylene oxide (PEO)-hyaluronic acid ... 阅读全帖 |
|
a**v
发帖数: 406 | 5 what kind of antibiotics?
aminoglycoside antibiotics (such as streptomycin, neomycin, kanamycin) will
kill hair cells in the inner cells resulting in hearing loss.
It's difficult (almost impossible) to recover the hearing. |
|
m******5
发帖数: 1383 | 6 I am currently using Z-competant sensitive DH5alpha cell as host strain for
plasmid construction using kanamycine containing plasmid.
There the protocol attached to the commercial kit mentioning that for kana-
resist plasmid transformation, an outgrowth in SOB medium is necessary, But
for convenience I am using LB instead . Does that really matter? Things do
not work smoothly, I am trapped in trouble shooting. |
|
l*********1
发帖数: 351 | 7 用pgex－4t－1表达gst－tagged 融合蛋白。pgex是amp抗性的，现在想把amp改成
kanamycin抗性的质粒。
是不是只需要剔除抗amp的TEM基因的orf片段，换成pet体系中的相关基因，保持原有
promoter即可？
谢谢 |
|
e**r
发帖数: 1144 | 8 1ng 质粒转化，只长了十几个
kanamycin浓度降到1/8， 没有明显改善
competant cell应该没有问题，用氨苄抗性的质粒测试应该在 10^8左右
有人遇到过类似现象没 ？ |
|
A********2
发帖数: 107 | 9 i) clone your target gene into a plasmid upstream of an antibiotic
resistance gene (e.x. Kanamycin) ii) amplify the two-gene cassette using
primers with 40nt homologous sequences iii) transform the linear fragments
into a recipient strain (e.x. E. coli) with an inducible Lamda Red System iv
) flip out the cassette with or without leaving a scar sequence, depending
on if there is FRT seq or I-sceI site in the cassette |
|
s******a
发帖数: 472 | 10 又来请教大家关于recombineering的问题了。因为重组是一个小概率事件（就算在重组
酶存在的情况下），所以就需要有筛选的策略。最简便高效的方法可以导入一个loxP-
Neo-loxP用于positive selection，最后可以利用cre重组酶去掉Neo。缺点是会留下一
个loxP位点，也不利于在该位点进行进一步的遗传操作。
鉴于此，我们决定采用positive/negative selection的方法进行recombineering。2种
常用方法包括galK筛选和rpsL-Kana筛选。我目前采用的是rpsL-Kana方法，Kana 使细
菌对Kanamycin有抗性，rpsL是细菌对streptomycin敏感。第一步recombineering
select “kana resistance”；第二步reombineering counter-select rpsL。
经过第一步，克隆PCR鉴定可以有95%正确率，即基本上所有克隆都带有重组的rpsL-
Kana。但是第二步（将rpsL-Kana替换为insert），获得阳性克隆如同中彩票一样，概
率很低。筛200... 阅读全帖 |
|
T**********t
发帖数: 1604 | 11 1）抗生素失效或浓度过低
2）污染
另外，阴性对照做了么？ |
|
|
b****r
发帖数: 17995 | 13 我做ampicillin倒是经常碰到这个问题
kana从来都是要有就是真家伙
估计是你的抗生素效价不行了，买新的吧 |
|
l**********1
发帖数: 5204 | 14 pVITRO1-neo-mcs
neo (kanamycin/G418 resistance).
pVITRO1-MCS
Expression plasmids pVITRO1-MCS are developed mainly for in vitro studies.
pVITRO1-MCS allow the ubiquitous and constitutive co-expression of two genes
of interest.
pVITRO1-MCS plasmids can be stably transfected in mammalian cells and the
genes of interest are expressed at high levels.
pVITRO1-MCS plasmids carry two elongation factor 1 alpha (EF-1α) promoters,
from rat and mouse origins combined to the CMV and SV40 enhancers
respective... 阅读全帖 |
|
d****i
发帖数: 2346 | 15 google搜索只看到有人这样配ampicilin。。。。。。。。。谢谢！ |
|
s******y
发帖数: 28562 | 16 You certainly can do it if you want to, but why do you want to do that? Why
not just water and then filter sterilize it? |
|
d****i
发帖数: 2346 | 17 这种配法放在冰箱不会冻住，仍然是液体，用的时候不用解冻，用完可以立即放回。。
。。 |
|
|
|
d****i
发帖数: 2346 | 20 谢谢大家！我查到kan在50%ethanol里面不溶解，已经配到水里面去了。 |
|
C******2
发帖数: 97 | 21
vectors
The construction is modified and derived from pRetrosuper which is used for
random mutation in host genome based on gene-trap. Truncated 3’LTR
fragment is promoter activaty lost. The idea is host genome will be mutated
by insertion because the existence of splicing acceptor(SA) fragment thus
generate a fusion RNA containing cherry CDS. Fusion florescence protein can
be detected in some case when insertion occurs as an in frame manner. The
insertion will also make the infected cells be... 阅读全帖 |
|
T****i
发帖数: 15191 | 22 只敢吃Ampicilin，但估计大多数细菌都有抗药性了。不敢吃kanamycin, antimycin什
么的。 |
|
c********n
发帖数: 225 | 23 谈谈和蛋白打交道的日子 （六）
引子
以前常和朋友谈起什么行业就业机会多，有长期稳定发展的潜力。翻来覆去后总结的是
，围绕着“生老病死，衣食住行”就靠谱。说到饮食，应个景，《舌尖上的中国》II谈
到了饮食与人文的关系，也用到了一些科学名词，比较煽情。而我比较喜欢的一档美国
cooking show，是Alton Brown在Food Network的“Good Eats”节目, 真正的touch了
一些美食背后的科学小常识, 做到了寓教于乐，fun。这篇就讲讲和食品相关的一些体
会吧。
第六篇 舌尖上的冒险
我们这里对于牛奶， 奶酪， 冰激凌等的要求是较高的。 对于我，尤其钟情冰激凌。
学校食品系研究生们的课题经常涉及各种口味和口感的冰激凌。他们常做的一个实验是
大众品尝评分， 就是给体验者每次几个相似的样品和一张问卷， 给各项指标（比如口
味，口感，甜度，等等）打分。 一般都安排在春季学期的尾巴和秋季学期的开始，大
约在午饭时间。做研究生的时候刚好在这个系的对面楼里， 每次都可以积极地参与，
经常午饭都省了。
由于大众需求，在食品系的一层，开了一个卖冰激凌的柜台，每过一阵子就有一种... 阅读全帖 |
|
