g***e
发帖数: 577 | 1 Comment: The fact that he'd be less physically intimidated by 100 whinnying,
duck-sized horses hardly matters.
Body:
President Obama's handlers failed to alert their boss to the most clever
question he was asked on Reddit in August: "Would you rather fight 100 duck-
sized horses or one horse-sized duck?" Staffers with more Reddit savvy could
've prepped an answer. Last autumn, New York Times columnist Nicholas
Kristof had a quick response. "Definitely one horse-sized duck," he typed. "
Then I'd ... 阅读全帖 |
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H********g
发帖数: 43926 | 2 https://www.technologynetworks.com/applied-sciences/news/from-poop-to-paper-
298888
It’s likely not the first thing you think of when you see elephant dung,
but this material turns out to be an excellent source of cellulose for paper
manufacturing in countries where trees are scarce, scientists report. And
in regions with plenty of farm animals such as cows, upcycling manure into
paper products could be a cheap and environmentally sound method to get rid
of this pervasive agricultural waste.
The... 阅读全帖 |
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N******t
发帖数: 16051 | 4
好吧，我犯懒了，抓了一句广告词
具体消化动物vs植物蛋白的机理，我还真不了解。兽医学方面的研究，似乎也都是面向
实践而不是机理
比如，他们讨论不同食物的 "digestibility" 和 "biological value"，基本就是看吃
进去的蛋白多少能被吸收，吸收的多少能被有效利用，这两个合起来又有 "net
utilization" 一说。这个貌似和蛋白来源，氨基酸成分，以及食物制作过程都有关。
有个研究比较了两个大众干粮和罐头：Morris, James G. and Quinton R. Rogers.
1994. Assessment of the nutritional adequacy of pet foods through the life
cycle. Journal of Nutrition 124:252OS-2534S，在这个网页也有讨论：http://maxshouse.com/feline_nutrition.htm，说是digestibility罐头90％，干粮80％；biological value罐头70%，干粮60%; net utiliza... 阅读全帖 |
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i*****i
发帖数: 2734 | 5 这个， 看上去还不错。第一个成分Chicken meal是什么东西？全鸡？：
Rocky Mountain Feline Formula
with Roasted Venison & Smoked Salmon
•For All Life Stages
•Grain-Free
•Roasted Venison & Smoked Salmon
•Optimal Amino Acids
•Digestive Support System
•Antioxidants
•Omega Fatty Acid Blend
•Berry Blend
•Taurine
•Tomatoes
•No Corn, No Soy
•Chelated Minerals
A grain-free formula for all life stages with peas and sweet potatoes
provides highly digestible ener... 阅读全帖 |
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x********u
发帖数: 15396 | 6 chicken feet works too
he pukes because he is not used to have "empty" stomach
he finishes digesting raw in 4 hrs also, while it takes around 10 hrs to
digest dry food |
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W********1
发帖数: 5019 | 7 【 以下文字转载自 GSD 俱乐部 】
发信人: Winner2011 (小崔), 信区: GSD
标 题: Foods Your Dog Should Never Eat
发信站: BBS 未名空间站 (Tue Apr 3 04:55:32 2012, 美东)
Dangerous Foods for Dogs
Who can resist those big brown eyes and cute doggie grin? Can a little
reward from the table really hurt your dog? Well, that depends on what it is
and what's in it. A chip with guacamole can cause your dog some real
problems. In fact, there's a lot of people food your dog should never eat.
And, it's not just because of weight. Some foods ... 阅读全帖 |
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R*********a
发帖数: 514 | 9 为什么carb多的猫粮不太好（长，欢迎校正/补充）
都是肉食动物，我们什么时候看到老虎啊狮子啊豹子啊，吃玉米，土豆，大米，大黄豆
，豌豆神马的。都是肉食动物，为什么要让猫儿吃呢？
小猫的生命只有一次，比我们短很多。我们都希望他们能健健康康，快快乐乐。
饮食是小猫健康情况最大的影响因素。所以健康平衡的饮食很重要。
不健康的饮食，会增加小猫生病的概率（肥胖，糖尿病，泌尿系统疾病等），即会让猫
儿和主人受很多不必要的肉体和精神的痛苦，也会增加主人的经济负担。
所以还不如，pay in advance, 尽量让小猫吃的健康一点点。
1.carb对猫体不好，会增加肾脏和泌尿系统疾病的概率。
2.carb不能提供猫体需要的营养。猫无法依靠谷物合成体内必需营养物质。
3.摄入大量carb容易产生饮食紊乱，抑制猫吃蛋白质，造成恶性循环。
4.carb对牙齿健康不好 （牙痛不是病，痛了真要命）
为什么商店买的干猫粮会有carb呢？
主要的原因是为了减低成本啊，成本低+卖的贵&多=多赚钱
另外，原料和成品相对易于保存
喂猫粮相对便宜，省事儿，但是考虑到可能产生的各种问题，
不如raw feeding或者罐... 阅读全帖 |
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K*******y
发帖数: 6003 | 10 还有一个药 Mirtazapine - 记下来
http://www.wisegeek.org/what-are-the-uses-of-mirtazapine-for-ca
In people, mirtazapine generally stimulates appetite. It also has an anti-
nausea effect, reducing vomiting. When cats take the medication, they often
experience these same benefits, so the drug can be useful when veterinarians
want to make sure animals keep food and water down, or when the goal is to
gain or maintain weight. A wide range of conditions can cause this need, but
kidney disease, pancreatitis an... 阅读全帖 |
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K*******y
发帖数: 6003 | 11 还有个药是这个，因为这个是人类的抗抑郁药，副作用就是瞌睡/口渴，不知道对猫来
说是不是也这样，我的猫吃了到不睡觉。我自己先尝了尝，不是很苦，很瞌睡（吃完就
坐电脑前睡着了），很渴。如果需要可以给你的猫试试。
我也不知道我的猫现在是受罪还是不受罪，她睡着的时候，看上去还好 :(
还有一个药 Mirtazapine - 记下来
http://www.wisegeek.org/what-are-the-uses-of-mirtazapine-for-ca
In people, mirtazapine generally stimulates appetite. It also has an anti-
nausea effect, reducing vomiting. When cats take the medication, they often
experience these same benefits, so the drug can be useful when veterinarians
want to make sure animals keep food and wate... 阅读全帖 |
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x******n
发帖数: 8550 | 12 交作业P78 49-53
49 梁昌年，吴雅楠， 鲍敏琪(p78) by xianyunn
无疑，基金会是一件很有意义的事，其宗旨是非常神圣的。这是我们的出发点与信心所
在……
－－梁昌年，SOSCEF会员（弗吉尼亚理工学院）
Undoubtedly, our Foundation has engaged in an immensely meaningful endeavor,
and its aim is a sacred one. These are where our starting point and
confidence lie.
- Changnian Liang, Member of SOSCEF (Virginia Tech)
◇记得在基金会成立之初，会员们曾经为给基金会取个合适的名字争论了好半天。后来
经过投票，终于决定取了现在这个名字。中国的教育真的危机到需要“救援”这个地步
吗？而且需要象我们这样一些身在海外的学生或工作了的人，在紧张的学习或工作当中
为此忙来忙去吗？说实在的，我有时也会这样怀疑地问自己。但不时地，从过去老师的
来信，从父母的来信和从国内... 阅读全帖 |
|
f*******p
发帖数: 521 | 13 a question: 《华枫园》or 《枫华园》? I tried to search 《华枫园》 to see if
there was formal English name for it but I found only 《枫华园》
47 中国教育救援基金会简介(p76)
原文：
朝花夕拾
基金会最早的报道见诸1993年出版的《华夏文摘－救援中国基础教育专刊》和《华枫园
》。本篇记录了十九年前创会义工的心声。倾听历史的回响，言犹在耳，心手相传....
..
我们没有忘记
中国教育救援基金会简介
吴雅楠
打开中国的地图，人们会感到那片古老的土地如今正在一片商业大潮中热烈地涌动着。
一向勤劳俭朴的中国人，现在抓住这千载难逢的机会，想使自己尽快脱贫致富。于是，
人们从报道中读到：万元户不稀罕了，南方的人们开始买房子、买汽车了，手中富裕的
人们又因炒股票而亢奋起来……那块贫瘠的土地一下子给人一种耀眼的色彩。
然而，在这种眩目的色彩背后却有着另一幅与此极不相称的画面：一直生活清苦而又任
劳任怨的中小学老师们并没有在这种大潮中得到他们应有的报酬，反而因通货膨胀而不
得不背起更加沉重的... 阅读全帖 |
|
x******n
发帖数: 8550 | 14 交作业P78 49-53
49 梁昌年，吴雅楠， 鲍敏琪(p78) by xianyunn
无疑，基金会是一件很有意义的事，其宗旨是非常神圣的。这是我们的出发点与信心所
在……
－－梁昌年，SOSCEF会员（弗吉尼亚理工学院）
Undoubtedly, our Foundation has engaged in an immensely meaningful endeavor,
and its aim is a sacred one. These are where our starting point and
confidence lie.
- Changnian Liang, Member of SOSCEF (Virginia Tech)
◇记得在基金会成立之初，会员们曾经为给基金会取个合适的名字争论了好半天。后来
经过投票，终于决定取了现在这个名字。中国的教育真的危机到需要“救援”这个地步
吗？而且需要象我们这样一些身在海外的学生或工作了的人，在紧张的学习或工作当中
为此忙来忙去吗？说实在的，我有时也会这样怀疑地问自己。但不时地，从过去老师的
来信，从父母的来信和从国内... 阅读全帖 |
|
f*******p
发帖数: 521 | 15 a question: 《华枫园》or 《枫华园》? I tried to search 《华枫园》 to see if
there was formal English name for it but I found only 《枫华园》
47 中国教育救援基金会简介(p76)
原文：
朝花夕拾
基金会最早的报道见诸1993年出版的《华夏文摘－救援中国基础教育专刊》和《华枫园
》。本篇记录了十九年前创会义工的心声。倾听历史的回响，言犹在耳，心手相传....
..
我们没有忘记
中国教育救援基金会简介
吴雅楠
打开中国的地图，人们会感到那片古老的土地如今正在一片商业大潮中热烈地涌动着。
一向勤劳俭朴的中国人，现在抓住这千载难逢的机会，想使自己尽快脱贫致富。于是，
人们从报道中读到：万元户不稀罕了，南方的人们开始买房子、买汽车了，手中富裕的
人们又因炒股票而亢奋起来……那块贫瘠的土地一下子给人一种耀眼的色彩。
然而，在这种眩目的色彩背后却有着另一幅与此极不相称的画面：一直生活清苦而又任
劳任怨的中小学老师们并没有在这种大潮中得到他们应有的报酬，反而因通货膨胀而不
得不背起更加沉重的... 阅读全帖 |
|
N*******8
发帖数: 1449 | 16 Science News
Appendix Isn't Useless At All: It's A Safe House For Good Bacteria
http://www.sciencedaily.com/releases/2007/10/071008102334.htm
(想看论文原文的去这里找:
Biofilms in the large bowel suggest an apparent function of the human
vermiform appendix Original Research Article
Journal of Theoretical Biology, Volume 249, Issue 4, 21 December 2007, Pages
826-831).
Science Daily (Oct. 8, 2007) — Long denigrated as vestigial or useless, the
appendix now appears to have a reason to be – as a "safe house" fo... 阅读全帖 |
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s*********t
发帖数: 4253 | 17 因为原文是希伯莱语，古时说的“鸟” 和 400百多年前人类定下“鸟”的
classification 是不一样啊。在那时“鸟”是指更上一层次的是会飞的动物。
兔子反刍。你读以下的，首先要了解希伯莱语原文.去查去google
Hebrew phrase for ‘chew the cud’ simply means ‘raising up what has been
swallowed’
不能用今天的定义套在古代的文字写法啊。
你读以下的：
In modern English, animals that ‘chew the cud’ are called ruminants. They
hardly chew their food when first eaten, but swallow it into a special
stomach where the food is partially digested. Then it is regurgitated,
chewed again, and swallowed into a different stomach. Animals ... 阅读全帖 |
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s*********t
发帖数: 4253 | 18 我查了下。这里有比较好的解释。（有时间读看。。）
http://www.apologeticspress.org/APContent.aspx?category=13&arti
Q.
The Bible speaks of two animals, the coney and the hare, as “chewing the
cud.” Isn't the Bible mistaken on this point? These animals do not actually
chew the cud, do they?
A.
once wrote: “Something that has long perplexed me is the way
that inerrancy proponents can so easily find ‘scientific foreknowledge’ in
obscurely worded Bible passages but seem completely unable to see
scientific error in statements ... 阅读全帖 |
|
s*********t
发帖数: 4253 | 19 因为原文是希伯莱语，古时说的“鸟” 和 400百多年前人类定下“鸟”的
classification 是不一样啊。在那时“鸟”是指更上一层次的是会飞的动物。
兔子反刍。你读以下的，首先要了解希伯莱语原文.去查去google
Hebrew phrase for ‘chew the cud’ simply means ‘raising up what has been
swallowed’
不能用今天的定义套在古代的文字写法啊。
你读以下的：
In modern English, animals that ‘chew the cud’ are called ruminants. They
hardly chew their food when first eaten, but swallow it into a special
stomach where the food is partially digested. Then it is regurgitated,
chewed again, and swallowed into a different stomach. Animals ... 阅读全帖 |
|
s*********t
发帖数: 4253 | 20 我查了下。这里有比较好的解释。（有时间读看。。）
http://www.apologeticspress.org/APContent.aspx?category=13&arti
Q.
The Bible speaks of two animals, the coney and the hare, as “chewing the
cud.” Isn't the Bible mistaken on this point? These animals do not actually
chew the cud, do they?
A.
once wrote: “Something that has long perplexed me is the way
that inerrancy proponents can so easily find ‘scientific foreknowledge’ in
obscurely worded Bible passages but seem completely unable to see
scientific error in statements ... 阅读全帖 |
|
s****a
发帖数: 39 | 21 【 以下文字转载自 Biology 讨论区 】
【 原文由 scilla 所发表 】
i run some eletrophoresis to see how the enzyme works on the plasmid DNA.
but something weird happen.
the control group (without digest enzyme) got 2 bands appear on the gel image
the digest group( all the 3 enzymes) got only 1 bands appear on the gel image
anyone has experience on this before?
anyone can give out any possible analysis on this?
i am just new to the lab, and my professor has no time to take care of me .
faint |
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u*****t
发帖数: 292 | 22
image
image
That's possible.
THe plasmid without digestion will generally have 3 bands based on the plasmid
configureation: the fastest one is supercoiled, the medium is linear ( both
strands of the plamsid were broken during extraction, and the slowest one is
the circular(one strand was broken). The quality determines the band pattern
in UNCUT plasmid.
You said only one band shows in 3-enzyme digestion. What is the size? You may
got four 2kb bands from a 8kb plasmid. BUT the most possible caus |
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T**********e
发帖数: 29576 | 23 下面这篇文章对Jobs的胰腺内分泌癌治疗总结的不错，看来死的有点冤。
1. 10-2003 确诊，07-2004终于手术切除，期间Jobs一直在试自己选的diet疗法。扫描发现癌变在扩大才动了手术，这段时间癌转移的多少不可知，但肯定越拖转移的越多。
2.手术后没化疗和放疗。Jobs 2005年演讲说这表明病情乐观，不知是哪个庸医给他灌输的。2004年手术是大面积切除，部分胰腺和胃和小肠，胆和胆管都切除，目的是要消除扩散，但不系统化疗和放疗怎么能最大程度减少扩散威胁？哪个外科大夫这么信自己的手艺？
3.2009年换肝，这在扩散性癌治疗是有争议的，因为换肝后压制免疫反应有可能促进别处癌的生长。也有可能当时肝已经不行了没办法。
这种癌正常治疗大多数可活10年以上，Jobs真该到东岸Hopkins或Harvard找个靠谱的大夫。
Jobs’s Unorthodox Treatment
Some of Apple trailblazer Steve Jobs’s cancer treatment choices, from
alternative therapies to a liver tra... 阅读全帖 |
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j*a
发帖数: 14423 | 24 import hashlib
m = hashlib.md5()
s='
6165300e87a79a55f7c60bd034febd0b6503cf04854f709efb0fc034874c9c652f94cc4015a1
2deb5c15f4a3490786bb6d658673a4341f7d8fd75920efd18d5a'
for i in [i*8 for i in range(16)]:
for j in range(4):
m.update(struct.pack('B',int(s[i+6-j*2:i+6-j*2+2],16)))
m.digest()
m = hashlib.md5()
s='
6165300e87a79a55f7c60bd034febd0b6503cf04854f749efb0fc034874c9c652f94cc4015a1
2debdc15f4a3490786bb6d658673a4341f7d8fd75920efd18d5a'
for i in [i*8 for i in range(16)]:
for j in range(... 阅读全帖 |
|
t**********s
发帖数: 930 | 25 只找到了一个 catalina.properties:
# Licensed to the Apache Software Foundation (ASF) under one or more
# contributor license agreements. See the NOTICE file distributed with
# this work for additional information regarding copyright ownership.
# The ASF licenses this file to You under the Apache License, Version 2.0
# (the "License"); you may not use this file except in compliance with
# the License. You may obtain a copy of the License at
#
# http://www.apache.org/licenses/LICENSE-2.0
#
# Unles... 阅读全帖 |
|
|
b*********t
发帖数: 65 | 27 小人才潜，只是知道个大概，PROTEOMICS是指
用分析的手段来表征一个生物体(或一种CELL)里的
所有蛋白。
现在作的最好的都是用MS的方法。也有尝试用
PROTEIN CHIPS的，不过还无法与MS的方法竞争。
一般MS用的是先DIGEST，然后在用MS(一般是MALDI)
这叫BOTTOM UP，方法很成熟，AUTOMATION的很好，
在这里又分2D-GEL和2D-LC/MS
AEBERSOLD R和YATES JR都是这种牛人
令一类的是想作完整的蛋白，即偶们老板的所谓的
TOP-DOWN方法，基本上是不用DIGESTION。现在这方面的
PAPER还不多，影响也不大。 |
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y***n
发帖数: 99 | 28
================
this is not right.
pI determined by the 2D gel system, the time you cut out the spot.
quatuple mass spec can actually sequence some partial sequence,
which is easy part. Hard part is that you cann't get enough protein
when go to the quatuple, Electrospray requires ten teims more protein
than MALDI.
In-gel digestion coupled by MALDI can give the masses of digested peptide.
use those peptide mass, there are websites you can search the potential
protein candidate with predited m |
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m****g
发帖数: 13 | 29 1. buy enough amount of commercially available herring (or salmon) sperm DNA.
They are dirty cheap. you can get 100mg/$200 probably anywhere.
2. digest your DNA using AluI restriction enzyme. If AluI does not work
well, try other enzymes that recognize 4-nucleotide palingdromic sequence.
You may combine 2 enzymes in one digestion mixture.
3. run a 6% denatured sequencing gel using a thicker spacer set (like 1mm), or
better (maybe) a 1.5% agarose gel to seperate your DNA.
4. Cut out y |
|
n*******e
发帖数: 27 | 30 use NEBuffer2+BSA, digest enough amount of PCR product O/N. i do not know
if you add some nucleotides for buffer purpose. if it is for mutagenesis,
or expression of certain gene, maybe difficult. however, check with BioLab
for Hind3, even 12bp sequence (6bp as buffer flanking) could not be completely
digested O/N. for Xho, it is better, about 75%. so the 1st thing you should
do is to check your design, order new primers if necessary. they are so
cheap nowadays. actually, since it is only 150bp. |
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s*******e
发帖数: 23 | 31 (1)
fusiong protein with a enzyme digestion site. overexpress it and digest.
check media by western.
(2)
EGFP fused to N tel. microscopy
(3)
homology search of the N-ter from database.
(4)
I don't know. :) |
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s******y
发帖数: 28562 | 32
Search the amino acid sequence on data base for signal peptide.
MOst signal peptides have been characterized.
I think, roughly, but not in detail: digest the whole cell or the membrane
vesicles make from supersonic, without using any detergent. If a pepetide is
outside membrane, it will be digested. If it's inside, it will not.
GFlycosylation should use similar mechanism, but instead of protease,
glycosylase is used to labled the outside peptide, then a mess spec can be
used to determined the l |
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r******m
发帖数: 173 | 33 I am trying to clone a 300 bp gene into pTYB2 vector (7.4kb)for later
protein purification method by IMPACT-CN. NdeI (producing sticky end) and
SmaI (blunt end) restriction sites were constructed into primers
corresponding to the N- and C- termini of the gene and incorporated by PCR
amplification. The PCR product and the vector were both double digested with
SmaI and NdeI respectively. After gel purification, the digested insert (
gene) and the vector were used to perform ligation with T4 DNA li |
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d******f
发帖数: 14 | 34 现在需要以下这篇文章，哪位大侠有这篇文章的请发到我的邮箱：brentchen1@hotmail
.com 万分感谢！
The ISME Journal 3, 700-714 (June 2009) | doi:10.1038/ismej.2009.2
Towards the definition of a core of microorganisms involved in anaerobic
digestion of sludge: Comparison of anaerobic digester populations |
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g*******q
发帖数: 15 | 35 For EcoR I and Xba I, "Double digest is not recommended. A sequential digest
is required, using each enzyme in its supplied NEBuffer."--from NEWB
website. |
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I*****y
发帖数: 6402 | 36 use trypsin to digest your protein overnight, and the amount of trypsin
needs to be higher to digest your protein into individual amino acids. my $0
.02 |
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e****s
发帖数: 1125 | 37 有没有做digestion test?
同时做一下single 和double digestion test,看看酶切位点对不对，Size对不对。 |
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z****g
发帖数: 3340 | 38 想找一个人的细胞表面分子marker能抗trypsin digestion 或对trypsin digestion不
太敏感。搜了半天没有任何有用的信
息。 |
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b******r
发帖数: 111 | 39 Anyone has experience of inserting 9kb in a 8.4kb vector? 2 months was gone
,but I get nothing. Would
you like to take a look a this protocol and give me some suggestion ?
Thanks a lot
Purpose: Insert 9kb mouse sequence into vector pPNT6.
Vector pPNT6: Its map is at http://www.addgene.org/pgvec1?
f=c&identifier=11072&atqx=pnt6&cmd=findpl . I have inserted 0.9kb DNA in
this vector at XhoI/EcorI
double sites. I should have no problem in cloning short fragment.
Insert DNA is 9kb.I have cloned t... 阅读全帖 |
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n********k
发帖数: 2818 | 40 谢谢你的详细意见。首先要说，我做分子克隆的经验不是很多。其次，基本不会不改变
条件反复重复。
1 当初选用pQE8也是没有办法，因为pQE9我们实验室没有（pQE8和9的区别就是8在酶切
位点之间只有一个碱基，而9要长得多）。最开始的时候没有意识如此近的酶切位点很
难切下来，最起码同时酶切不可能切下来。但是只尝试过一次顺序酶切不行就没有再尝
试用pQE8了，我不记得是先切BamH1还是Hind3，后面尝试插一段片段在2个酶切位点之
间再切。
Nice alternative, shall solve the problem in this case...but I would just do
blunt ligation, nowadays, the fast ligation kits from different companies
such as Roche, takara, don't really care about whether it is sticky or blunt
ends, it works just as well...BTW, any one else ... 阅读全帖 |
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a***e
发帖数: 1010 | 41 depends how you do SNP mapping after that.
for us, just digest sample with proteinase K, then denature proteinase K,
then PCR, then restriction digestion. that's good enough. |
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D*a
发帖数: 6830 | 42 Quick Tail Prep
Collect mice figure/tail in 0.5 ml Eppendorf tube. (4 mm is enough)
Add 100 microliter of quick tail digestion buffer (below).
Incubate at 55 C for 2 hours to overnight.
Vortex and incubate at 95 C for 10 minutes.
Centrifuge for 5 minutes to get rid of the small pieces of undigested sample.
(NOTE: I do neither vortex nor centifuge, and my pcr works
but if you vortex, you have to centifuge)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever ... 阅读全帖 |
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s******y
发帖数: 28562 | 43 哦，这样的啊。
如果是培养的细胞的话，一般需要洗一洗来去掉培养基免得在分析样品的时候带进污染。
如果是在小虫里收集的胃。。。会不会有体液或者食物污染？如果有还是
要洗洗的。如果没有这个顾虑那就别洗了，听你讲起来好像样品很小的样子，
弄不好一洗就找不回来了。
如果裂解液里面的detergent过多，会干扰TCA沉淀的效果的。不过如果detergent
在１％以下还是可以用TCA的。TCA沉淀的时候蛋白都变性了，不会有phosphatase 作用。但是在裂解的时候，如果是用detergent而不是Urea or guanidinium 的话，phosphatase 会是一个大顾虑。可以用Sodium Orthovanadate (Vanadate, Na 3 VO 4)来抑制phosphatase.
如果用8M Urea，一般都是8M Urea in PBS, pH 大致调一下不要超过９就好了。
1 mg 组织? 这么少？一般而言20倍体积8M Urea就够了，所以20ul 就够了，
短暂的超声一下(15~30sec)，然后vortex 一分钟应该就好了，最后再加1/4 的 5X SDS... 阅读全帖 |
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W*********n
发帖数: 775 | 44 please help me to have a look at the whole protocols and give some
suggesitons:
1) get 50midguts (about 3000cells/midgut, so about 1,500,000 cells or 80ug
protein totally),add 80ul lysis buffer (8M Urea, in 1XPBS, pH=8) including
Protease inhibitor and phosphatase, ultrasonic for 15-30 sec, then votex for
1 min. centrifuge for 10min at 14,000g and take the supernatant, add 16ul
5X loading buffer and heat for 20--30 min at 60 degree. load gel at 30ul/
lane and run for 2cm(for each sample, run th... 阅读全帖 |
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W*********n
发帖数: 775 | 45 谢谢艳阳天！
和我们这边作质谱的老师交流，问他用什么样的裂解液最适合后面的LCMSMS分析，他说
“Since you’re running the samples on a gel before trypsin digest, don’t
worry about the buffers; use whatever you want. ” 然后我就把上面那封邮件发
给他，他回复“I don’t think urea is a good idea, since it will cause
problems for your gel (very difficult to totally remove). I would be
careful during your gel digest…make sure you do plenty of washes to remove
the protease inhibitors before the trypsin step. ”
我晕死了，他都说use whatever you want了，我找了大家认为很好的Urea方法，结果
他又说not... 阅读全帖 |
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s*******s
发帖数: 623 | 46 谁叫人家是名人呢～
http://www.ncbi.nlm.nih.gov/pubmed/22123987
Proc Natl Acad Sci U S A. 2011 Nov 28. [Epub ahead of print]
Synthesis of three advanced biofuels from ionic liquid-pretreated
switchgrass using engineered Escherichia coli.
Bokinsky G, Peralta-Yahya PP, George A, Holmes BM, Steen EJ, Dietrich J,
Soon Lee T, Tullman-Ercek D, Voigt CA, Simmons BA, Keasling JD.
Source
Joint BioEnergy Institute, 5885 Hollis Avenue, Emeryville, CA 94608.
Abstract
One approach to reducing the costs of advanced bio... 阅读全帖 |
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H****N
发帖数: 997 | 48 He/she digested the DNA first before heating to 65 degrees. Also, there is
no smear in the other digestions with different REs, which is clearly against
your argument about DNase. DNAse would make the fragments smaller, not bigger. |
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a*****a
发帖数: 415 | 49 fermentas的酶没用过
neb的用 neb double digest finder查
ecori和bamhi如果都是HFversion的可以用buffer 4做double digest
bamhi如果不是hf version最好在buffer3切，不然star activity强 |
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s******a
发帖数: 472 | 50 I used Amersham Direct Labeling Kit for my southern blot, and the protocol
is basically the kit manual. However, I have now a reproducible problem. As
shown in the blot, I expect a specific band in digested genomic DNA, but all
I have is a big smear (inside which there seems to be a band higher than my
target band).
I might have at least two problems:
1, the probe is bad;
2, the digestion is incomplete;
I kindly ask you to give me some advice regarding trouble shooting and how
should I proceed.
... 阅读全帖 |
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