q*****n
发帖数: 6 | 1 there are about 10 gene between two flanking marker. I did the RT-PCR and
qPCR to check the expression of candidate gene bewteen the mutant and wild
type.
your |
|
o******n
发帖数: 511 | 2 背景：
1. 有个酶能分解一种底物，其产物可作为细菌的唯一氮源。这个酶连带它上下游
flanking region一起放到质粒上，转到大肠杆菌里，大肠杆菌可以表达这种酶，别人
测过cell-free extract里的酶有活性。
2. 已知这种酶Km比较低，对底物亲和力低
3. 土壤细菌本身有这个酶的，在minimal media+底物的平板和液态培养基都可以长起
来，长得慢。
4. 大肠杆菌好像不太喜欢这个6 kb多的质粒，每次overnight culture都长得不太浓，
有floc,显微镜下看到细胞连成长条，肉眼可见细胞沉在管底。
现在我在minimal media+底物的液态培养基里想长带这个酶的质粒的大肠杆菌，就是长
不起来，我试了几种minimal media，加了trace elements，也不行。大肠杆菌在
minimal media加别的氮源可以长。
因为酶不给力吗？可是再怎么不至于一点都长不起来呀？换个很强的promoter，多产些
酶有用吗？
还是因为酶在质粒上，而大肠杆菌不喜欢这个质粒，在minimal media里，就不爱给表
达它了？
请大家给些建议意见... 阅读全帖 |
|
a********n
发帖数: 844 | 3 我研究的转录因子不是这个，不排除有相似的机理。首先，不排除两个转录因子调控同
一个基因，但具有不同的的结合能力和调控力度，这有可能是DNA element在被调控基
因里并不是perfect，flanking sequence等因素（即某个基因的promoter序列）共同决
定转录因子的结合。其次，两个转录因子是不是同时表达在同一个细胞里边。 |
|
R****n
发帖数: 708 | 4 you are talking about Methylation Specific-HRM, I assume. Althought you are
using qPCR system, you didn't use a beckman probe to identify the sequence
change. So this is not a qPCR method.
ZymoResearch's kit should be fine. We used their kit a lot. There are some
commercial M or UM DNA standard which have been converted. You can use them
to test your primers.
How do you design your primers? flanking any CpG sites?
If you didn't change primer, I guess that you may have PCR product
contamination i... 阅读全帖 |
|
t****t
发帖数: 610 | 5 Thanks. It's Methylation Specific-HRM. I'm studying the same CpG islands,
so primers are all the same.
Primers do flank CpG sites.My primers work.
Even I have PCR product contamination, how it is possible to get a Tm lower
than unmethylated control? I have PCR controls, which are already converted
standard DNA (methy and unmethy). They were fine with the right Tms.
Also, although I'm using the same reagents (kit, buffer,...), last week
there was once that my results were fine. Really got confus... 阅读全帖 |
|
g***j
发帖数: 40861 | 6 Tough Lessons From Golden Rice
Martin Enserink
It was supposed to prevent blindness and death from vitamin A deficiency
in millions of children. But almost a decade after its invention, golden
rice is still stuck in the lab
It's easy to recognize Ingo Potrykus at the train station in Basel,
Switzerland. Quietly waiting while hurried travelers zip by, he is holding,
as he promised, the framed and slightly yellowed cover of the 31 July 2000
issue of Time magazine. It features Potrykus's bearde... 阅读全帖 |
|
m******5
发帖数: 1383 | 7 specifically which kit from Ambion?
I knew the sequence, but I was not sure if we can directly add the sequence
in a 5' to 3' manner upstream of antisense primer, is there any requirement
in the regards of linker or flanking sequence? |
|
g*********d
发帖数: 233 | 8 The role of small non-coding RNAs in genome stability and chromatin
organization
http://jcs.biologists.org/content/123/11/1825.full
Josien C. van Wolfswinkel and René F. Ketting*
Journal of Cell Science 123, 1825-1839
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.061713
Summary
Small non-coding RNAs make up much of the RNA content of a cell and have
the potential to regulate gene expression on many different levels.
Initial discoveries in the 1990s and early 21st centur... 阅读全帖 |
|
y*********u
发帖数: 183 | 9 that is also how I figured it, I designed such a knock in animal by
replacing the whole exon1 and exon2 (keep some flanking sequence as splicing
context) without additional polyA signal. I am hoping it would work
binding |
|
b******s
发帖数: 1089 | 10 I tried to amply 2XGFP by one PCR and subclone it into some vectors. The
template contains two tandem GFPs but PCR can produce only one 700bp band.
The 1.4K band is very weak and smeared. Before redesigning long gateway
primers to include a little flanking regions, I want to see if anybody have
trick for such PCR? Thanks a lot! |
|
y*********u
发帖数: 183 | 11 如图
我要knock in表达的原基因有4个exon，我想插入一个EGFP reporter
，于是把exon1和exon2 replace掉（留下一小部分flanking sequence）
因为这个基因的3'UTR的调控做得很热，因此我就不能引入外源polyA 比如SV40, BGH
polyA等
粗看过去似乎问题不大，我用的EGFP的编码做过修改不含有splicing context
但是也有人说不加外源polyA的knock in表达很靠运气？ |
|
n********k
发帖数: 2818 | 12 never did cloning this way myself...but I advised my former roommate to
develop the tech and I remembered he inserted three LoxP sites into a 25kb
or so plasmid within literally 2-3 weeks...I could be wrong since I was not
the one doing it...but I thought the concept was simple: u have the homology
adapters flanking the sequence of interests...someone say 30 bp may be too
small and could get lost...never did myself, don't know(maybe I shouldn't
have commented so surely:)))...I could ask my form... 阅读全帖 |
|
C*******e
发帖数: 4348 | 13 lambda red system的有一个2002年的PNAS
那个实验室有这个E.coli菌株
插入片段两端有46nts的flanking region就可以了
线性DNA片段和目标plasmid一起电转化到那个E.coli菌株就行
就是你要有个方法筛
比如线性DNA片段上有另外一个抗性之类的
不然大海捞针 |
|
v***a
发帖数: 1242 | 14 抱歉没有讲清楚。情况是这样的：拿不同cDNA sample做模板，PCR一个gene（非全长）
，大部分的sample都能得到一个正确的产物短条带（经过测序验证）。然后想用PCR的
方法得到全长的该gene，设计flank该gene两端的引物后，却几乎得不到任何条带。做
到现在，试了自己的sample，也拿别人做的cDNA试（大约20多个来源不同的cDNA
sample)，只有2个是有条带的。用这2个purify后的PCR产物做transformation都失败了
，所以还需要尝试更多，但又苦于能PCR得到全长的概率很小。在此请教大家。 |
|
B******o
发帖数: 496 | 15 不太明白你的意思。就我知道的表达microRNA的方法，最常用的有两种，一种是pre-
miR再加上其flanking的genomic sequence。一般左右两边取150bp左右就足够了。另一
种是用已知的microRNA backbone，把中间的mature miR sequence给换成你想表达的
miR。这种方法好像就Hannon等人在用，不普遍。我自己的经验是，第一种方法比较好
，特异性好，表达量高。
用Genomic DNA做模板。cDNA没法做吧。primary transcript很快就被Drosha处理掉了
，你的cDNA里啥都找不到 |
|
v******l
发帖数: 235 | 16 Would someone please help me download this paper? I can't find in our
library. Thanks a lot! My e-mail: j********[email protected]
PMID: 21197627
http://www.ncbi.nlm.nih.gov/pubmed/21197627
Genet Epidemiol. 2010 Dec 31. [Epub ahead of print]
Predicting multiallelic genes using unphased and flanking single nucleotide
polymorphisms.
Li SS, Wang H, Smith A, Zhang B, Zhang XC, Schoch G, Geraghty D, Hansen JA,
Zhao LP.
Source
Division of Public Health Sciences, Fred Hutchinson Cancer Research Center,
Seatt... 阅读全帖 |
|
l**********1
发帖数: 5204 | 17 你老板没让你事先设计 reporter+ cassette modified vector 就做plasmid then
BAC transgene to Mouse 啦?
then 要证明Your transgene（insert）整合到特定locus
if yes plus next Long-range PCR does not work well.
return to start point then do below steps or similar steps:
Generation of knockin mice
Bacterial artificial chromosome (BAC) clone RP23-135A18
containing mouse genomic DNA encoding exon 1 of Zeb1 was
digested with PacI/SphI and SphI to yield 7.6-kb and 2.6-kb
homology arms, respectively, for targeting constructs (Figur... 阅读全帖 |
|
y*********u
发帖数: 183 | 18 问问，这种transgenic mice常用组件质粒哪里有卖啊？
搜了半天搜出来一堆老鼠，没有质粒 |
|
v*********d
发帖数: 382 | 19 jackson has iDTR mice available, for plasmid, you has to request from
Germany |
|
n******u
发帖数: 418 | 20 同源重组也是有长度限制的吧？
有些比较大的gene，经常看到也就是flank在某几个exon的边上，而不是全部替换掉 |
|
x******a
发帖数: 143 | 21 Anybody working with Drosophila FlyC31 has experience of dropping the RFP
flanked by loxP using Cre line?
I've done some crosses and have really weird problems.
Many Thanks in advance!!! |
|
|
b******y
发帖数: 627 | 23 If affecting the folding of flanking regions is your only concern, just use
a flexible linker.
On the contrary, I would worry about folding if you literally link two
compact domains without any linker. For instance, domain A ends with a helix
and domain B starts with a beta-strand. If you create an A-B polypeptide,
it is conceivable the last helix of A induces the first beta-strand into a
helix and therefore destabilizes the core of domain B. |
|
m******5
发帖数: 1383 | 24 如图，有一个BAC transgenic Vector含有一个要命的FRT flanked Neo cassette
必须用flipase切除
为省时间，想co-microinject一个CAGGS-Flpe plasmid
没有查到相关文献，但觉理论上可行，求高手re-assurance!! |
|
r*******y
发帖数: 48 | 25 1. floxed 300 bp should not present any problem for recombination;
2. introducing a disgestion site is a good and reasonable design for future
southern verification, if there is no endogenous restriction site(s)
available for southern strategy;
3. you should pay more attention to the design of the arm length. Based on
my experience, over 5000bp/arm is recommended; shorter than this will reduce
the specific recombination efficiency, although some reports also suggested
that a shorter arm also wor... 阅读全帖 |
|
l**********1
发帖数: 5204 | 26 RE LZ:
pls try
Cassette-ligation downstream 3'RACE PCR
or Tail-PCR
Protocol:
看图说话
>
Amplification of FSTs by 3’-RACE
Total RNA was isolated using hot-phenol extraction
[27], from 20 ml cultures in 50 ml Falcon tubes on a rotating
wheel. Reverse transcription used the SuperScript
III First-Strand kit from Invitrogen in a DNA Engine
PCR machine (MJ research). Primer QT was mixed with
5 μg RNA, incubated at 65 °C for 5 min then cooled to
55 °C over 100 sec, after which the annealed mixture
was kept... 阅读全帖 |
|
m******5
发帖数: 1383 | 27 如标题，这是第一个问题
第二个问题是，大家用在mES cell里通过转染Flpe表达载体的方式delete过FRT
flanked 区域么？ |
|
h**********r
发帖数: 671 | 28 Title:
Efficient homologous recombination with short length flanking fragments in
Ku70 deficient Yarrowia lipolytica strains
Journal
Biotechnology Letters December 2012
my email is m******[email protected], Thank you so much! One baozi will be sent to
you. |
|
z*t
发帖数: 863 | 29 问个问题哈，talen切割后不是通过引入的片段同源重组来edit genome么？
楼主若是相加个gfp在同时转针对特定序列的taln + 两侧用同源片段flank的GFP
plasimid就可以了？ |
|
z*t
发帖数: 863 | 30 问个问题哈，talen切割后不是通过引入的片段同源重组来edit genome么？
楼主若是相加个gfp在同时转针对特定序列的taln + 两侧用同源片段flank的GFP
plasimid就可以了？ |
|
s******r
发帖数: 1245 | 31 他的意思是他们在exon和intron中间插了个loxP，但是sequence出来的exon后面直接就
是intron，那个loxP不见了，看上去就和wt一样。扩4k很正常，保险点的PCR一般是要
flank homo arm的看当时es怎么做的了，当然southern啥的都做了，扩短一点可能问题
也不大。
建议楼主仔细查查这个knockin当时是怎么做的，那个位置是不是应该有这个loxP，这
个设计图看着很奇怪，是不是漏了点啥或者有地方画错了。另外可以用P1测一下看看能
不能测到GFP cassette，这个长度测序末尾应该能测到外源序列的，那就确信测得是
targeted allele了 |
|
a*******o
发帖数: 16 | 32 一般直接模仿miRNA在体内biogenesis的过程吧，在genome上找到pre-miR的sequence，
然后在5'和3’各加100bp左右的flank region， 这样貌似表达效率会比较高。 |
|
C*******e
发帖数: 4348 | 33 对于问题（1），就是你把自己最后要得到的质粒用手画也好，用软件也好，序列整出来
把inseert的部分highlight
就可以看的比较清楚了
决定好insert的部分以后，再往上游数个15－20bp（看手册怎么说）就可以
Gibson的是从5'端chew back
你可以选择保留质粒上的酶切位点（比如你这里的EcoRI）或者不保留
看你需要
所以如果保留位点就是
------G AATTinsert AATTC-------
------CTTAA insertTTAA G-------
如果不保留位点，就是这样：
------G insert （AATTC）-------
------C（TTAA） insert G-------
（括号里面因为5' chew back，而且insert上的flanking region不带这个“AATT”，
就失去了）
我一般还是选择保留酶切位点，这样的好处是如果这个片段我以后还想用来做别的
我... 阅读全帖 |
|
l**********1
发帖数: 5204 | 34 key words:
5fC; 5 caC; pre-mRNA splicing; Pol II; hetrochromatin or H4K20
cited from
Nat Struct Mol Biol. 19: 1061-4. (2012).
New functions for DNA modifications by TET-JBP.
http://www.ncbi.nlm.nih.gov/pubmed/23132381
>the ability of 5fC and 5caC to decrease the transcription elongation rate
of Pol >II may facilitate the interaction of Pol II with diverse
transcription elongation factors, chromatin regulators, histone-modifying
enzymes and factors involved in pre-mRNA splicing. Shukla et al.34... 阅读全帖 |
|
l**********1
发帖数: 5204 | 35 cited,
>Both zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs)
>can be used to mutagenize genomes at specific loci, but
these systems require two different DNA binding proteins
flanking a sequence of interest, each with a C-terminal
FokI nuclease module. As a result these methods have not
been widely adopted by the plant research community.
from
>Belhaj K et al., (2013).
>Plant genome editing made easy: targeted mutagenesis in model and crop
plants using the CRISPR/Cas >system.
>P... 阅读全帖 |
|
s******9
发帖数: 283 | 36 http://www.sciencemag.org/content/344/6179/24.full
Chasing the Money
Jennifer Couzin-Frankel
As constraints take hold in biomedicine, scientists are forced to adapt.
Money. It is what fuels research, and these days, it's almost all biomedical
scientists in the United States can talk about.
They've been buffeted by funding swings at the National Institutes of Health
(NIH), their field's primary benefactor. And now they're anxious about the
future, as Congress tries to rein in debt by slowing gove... 阅读全帖 |
|
g*********3
发帖数: 177 | 37 Hi All,
Recently I am doing CHIP-QPCR to test one TF binding. I use 10% of sample as
Input and the rest 90% to do IP. I have the TF-positive cell line A and TF-
depleted cell line B.
Primer is flanking the targeting site of TF.
The result I have is:
In A: Ct in IP 26 & Ct in Input 20 [Ct difference is 4]
In B: Ct in IP 30 & Ct in Input 20. [Ct difference is 10]
It seems TF binds to the targeting site.
However, I followed the calculation process of other people to estimate
yield:
1/9*1/(2^6)
whic... 阅读全帖 |
|
c*******e
发帖数: 5818 | 38 没太明白，我的entry DNA is PCR product，因为没有att LR，所以想再引物上加。你
这听起来象你的entry DNA已经有flank的att LR site，这样是不是就可以直接用
vector去做了。 |
|
h**********e
发帖数: 18 | 39 一个基因20个exon, 某个microdeletion包含了整个exon2, and 3.那么这个基因可能表
达吗？有transcript吗？
不明白为什么做mutation的时候flank一个exon整个蛋白都检测不到了？
谢谢！ |
|
x********e
发帖数: 35261 | 40 只是互补的话PCR的时候能成功吗，特别是flank两个exon的PCR？ |
|
w***r
发帖数: 709 | 41 这个人太牛X了。他在song h 甲基化文章真的很炫!！
但是我个人觉得
Cytochrome P450 drives a HIF-regulated behavioral response to reoxygenation
by C. elegans. Science (2013) Vol. 341 no. 6145 pp. 554-558. PMID: 23811225
这篇文章似乎新意不够，hif 和p450和缺氧的关系早就知道了。下面是一篇，还有不少
有明白人讲一讲吗？
Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated Cyp1a1
promoter activity by hypoxic agents.
Kim JE1, Sheen YY.
Author information
Abstract
Since hypoxia-inducible factor-1alpha (HIF-1alpha) and the arylhydrocarbon
receptor (AhR) shared th... 阅读全帖 |
|
w***r
发帖数: 709 | 42 这个人太牛X了。他在song h 甲基化文章真的很炫!！
但是我个人觉得
Cytochrome P450 drives a HIF-regulated behavioral response to reoxygenation
by C. elegans. Science (2013) Vol. 341 no. 6145 pp. 554-558. PMID: 23811225
这篇文章似乎新意不够，hif 和p450和缺氧的关系早就知道了。下面是一篇，还有不少
有明白人讲一讲吗？
Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated Cyp1a1
promoter activity by hypoxic agents.
Kim JE1, Sheen YY.
Author information
Abstract
Since hypoxia-inducible factor-1alpha (HIF-1alpha) and the arylhydrocarbon
receptor (AhR) shared th... 阅读全帖 |
|
Q*****G
发帖数: 638 | 43 想在目的基因的N端或C端knockin GFP
利用cas9n d10a 载体，分别克隆２ guild RNA。
Donor repair template 突变掉guild RNA 序列。５＇和３＇flank分别为８００bp左
右.转染（LTX／Fugene HD）完３天可以看见荧光.可是传代后荧光非常弱，还可以做细
胞分选么？
通常转染后多久细胞分选?
是否有更好的方法可以提高荧光的强度和crispr的效率？
谢谢 |
|
l********e
发帖数: 415 | 44 说明你的qPCR引物设计的相当之烂，因为正常人都是设计flanking intron，在DNA模板
上是拉不出东西来的。 |
|
D**F
发帖数: 54 | 45 有个转基因老鼠，不知道准确的插入位点，没办法pcr鉴定杂合还是纯和 |
|
w*********3
发帖数: 217 | 46 用关键词Hi-tail查文献。照着做就可以了。
★ 发自iPhone App: ChineseWeb 16 |
|
k****l
发帖数: 279 | 47 PCR 扩增 flanking sequence，测序
或者全基因组测序
random |
|
f******y
发帖数: 73 | 48 39. A 63-year-old man is brought to the emergency
department 3 hours after the acute onset of severe right-sided flank
pain. He has a 9-year history of gout. His blood pressure is 110/84
mm Hg, pulse is 78/min, and respirations are 16/min. Examination
shows normal bowel sounds and no abdominal tenderness or masses. Urinalysis
shows 40 erythrocytes/hpf. Intravenous pyelography confirms a
right ureteral calculus. Which of the following is the most likely
underlying mechanism of this patient's urol |
|
a**********0
发帖数: 87 | 49 Finally got it done. Was a real nice surprise, thought I was going to flank
it.
Applied and got scheduling permit in the end of May, could not schedule near
my home, decide to wait till
July, spoke with my chief resident, she gives me one month of ED rotation to
take my step 3.
materials used:
UW, did one time, 55%, took notes in first aid. (in april, may, starting
june, two weeks of orientation, then two
weeks of night float, did not read anything)
CCS UW, did all the simulated 44 (or 40?) case |
|
a**********0
发帖数: 87 | 50 some friend asked:
恭喜 done with Step 3 with a good score，请问你的CCS成绩是不是在high
performance那块？所以才导致NBME成绩和考分严重脱节？ 还有CCS的CASES是不是都
在UW上了？？？ 有多少多媒体题？MCQ是不是和UW很象呢？我也是没时间看书，要考
试很烦。
answer:
CCS is average, yes, almost all, some has little variation.
3-4 multi-media.
not really, a lot of questions that are not in UW, at least 5 derm question
per block, very wired.
flank
near
to |
|
