From AlleleBlog: http://allelebiotech.com/blogs/2010/09/congress-may-let-sbir-authority-lapse-this-week/
The SBIR/STTR/CPP now appears likely to expire on Thursday night, September 30.
Some will deny it but here’s what’s happening.
Allegedly the Senate and House were close to a compromise complete with an 8 year
reauthorization of SBIR/STTR/CPP but each time it goes back to the House (Nydia &
Day), they change the VC language to masquerade 100% VC involvement as a compromise.
Because time is so short, the Senate passed a bill (S.3839) to simply extend
SBIR/STTR/CPP through January 31, 2010. The House was going to pass it on Wednesday
with the President signing Thursday. However, the word on the street is that Nydia
Velazquez, chair of the House Small Business Committee, and her illustrious second,
Michael Day, are rejecting the bill and are poised to let SBIR expire if necessary,
at least in the short term.
It seems that Velazquez’s hope is to move the SBIR reauthorization into the lame
duck session and incorporate all her Wall Street investors’ 100% non-compromise VC
ownership and jumbo award support into a must pass, end of the year omnibus bill
that can’t be touched by her detractors.
This sounds like a script for TV, but several years ago we had a similar year end
omnibus situation involving Nydia (as ranking member) and Sam Graves (subcommittee
chair) and BIO/NVCA, but the main difference was that the small business committee
chair was Donald Manzullo who nipped it in the bud. In our scenario today we have
to look to the House leadership to do it, but it will take your involvement.
Many senior people in the democratic party called for the House to support the
Senate compromise bill H.R. 2965, but Nydia ignored those calls, as did Jason
Altmire, the creator of this infamous Altmire Quagmire. Now Nydia’s really “miffed”
because last week she tried to “scrub” H.R.5297, the Small Business Jobs Act of
2010, but the Obama administration and Speaker Pelosi rolled her over and passed it.
CALL TO ACTION
If SBIR is important to you and your company, it’s time to get serious and realize
that this program can, and will go away unless you make a big noise to let your
politician’s know how you feel. All of us are sick of this, and we’re now facing a
lapse. Eight times this program has been deemed important enough to keep going (via
a CR) but will Nydia be successful in blocking this ninth attempt?
Voting will occur in the House on Wednesday and this may be the last time until
after the election that the SBIR extension bill could voted on. That means we must
act on Tuesday, September 28.
Here are some suggestions and rationale behind them.
CALL CALL CALL the House Tuesday September 21! Call Nancy Pelosi’s office at (202)
225-4965, Steny Hoyer (majority leader) at (202) 225-4131, Nydia Velazquez (202)
225-2361, also the House Small Business Committee line (202) 225-4038
Those of you who are good democrats, call the remaining House Democratic caucus
leaders: John Larson 202- 225-2265, Xavier Becerra 202-225-6235, Jim Clyburn
(202)225-3315
Those of you who are good republicans, call John Boehner (202) 225-6205, Eric Cantor
202-225-2815
Tell them in your own words that SBIR is about to expire and is being held hostage
by Nydia Velazquez. Let them know how important continuation of SBIR is to your
business and the country. Ask them to please support S.3839 (additional temporary
extension of programs under the Small Business Act and the Small Business Investment
Act of 1958) to keep the program from lapsing this week.
I realize that I’m asking you to do something that requires a good chunk of your
time. However, at the risk of losing you as a reader I must tell you that I donate
a large share of my time to try and keep you informed about this program, and I’m
not asking you to do anything for me, only for you and others like you. We do have
some good representatives from both parties BUT they need to hear from you and
quickly.
If you’re bold ask, “I would like to know how a party can let itself get hijacked by
a few people (like Nydia) on a vitally important, highly regarded and accepted
program. This action is to the detriment of your constituents, the country, and
yes, even your own party!”
Here’s what’s going on in the back rooms (formerly smoke filled) The Senate agreed
on a 4 month extension for SBIR because they (Senate) largely (including many on the
Republican side) did not feel a reasonable bill could be passed in the lame duck
session. The Senate has offered up some huge compromises that some believe even
James Greenwood from BIO could live with. The very long shot is that with enough
pressure we might get a compromise bill passed by Thursday.
WHAT HAPPENS IF SBIR LAPSES, EVEN FOR A SHORT TIME
This is an interesting question. Theoretically those projects (grants and
contracts) that are already in place should be okay, but some not. All new unsigned
agreements would stop. Agency comptrollers may start adjusting their budgets to put
the overall 2.8% SBIR/STTR back into their own research pools. Administrative
funding for SBIR could be severely cut back. Remember, all of your grants and
contracts are “subject to the availability of funding.”
On the other hand, SBIR can be voluntary, so some agencies may choose to keep their
SBIR doors open, hoping for, or expecting the reinstatement of the program.
In any event, this is bad for you and the agencies.
The Insider will be on the Hill Wednesday and Thursday, so we’ll do a follow up
report to you asap.
Rick Shindell
SBIR Gateway
Zyn Systems
40 Alderwood Dr.
Sequim, WA 98382
360-681-4123
rick@zyn.com
www.zyn.com/sbir
New Product of the Week 092810-100310:
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Promotion of the Week 092810-100310:
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Wednesday, September 29, 2010
Sunday, September 26, 2010
Dealing with Interferon Response When Doing RNAi
From AlleleBlog: http://allelebiotech.com/blogs/2010/09/dealing-with-interferon-response-when-doing-rnai/
Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.
Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.
Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.
Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.
Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.
To read the wholr blog, click here.
Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.
Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.
Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.
Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.
Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.
To read the wholr blog, click here.
Sunday, September 19, 2010
韩寒: 游行的意义
在jiu月18日这个敏感的时刻，我有的朋友开始研究要不要you行。当然，游的主体可以是反ri保diao救船长。终于，在一个很多论坛里连“you 行”两个字都打不出来的国家里，我们有行可以游了。那么，要不要参加这次命题一日游呢？
首先，我认为在现代中国社会中，分为三个阶级，那就是主子，奴才和狗，而我们往往一人饰两角，至于饰演哪两个角色，我想不会有人觉得他在演主子吧。前一阵子，主子需要奴才去附和和伺候，但是现如今，主子需要狗去吼两声，因为在狗的逻辑里，无论主子怎么对待它，只要有外人来犯，狗总是该看家护院的。
当弄明白了这个以后，回头想想就容易多了。但是，在这三个阶级以内，好在我还有选择做花花草草的权力。我的选择依据是，对于相关部门，小事和大事他们的区别就是抗议一次和抗议十一次，有特权有能力的地方尚未出力，除了把人家日本大使变成了应召男郎以外，我们相关部门情绪稳定，并不见什么实际决心，别说武力上，连经济上都不敢有所动作。他们韬光养晦，所以我也韬光养晦。毕竟，我等做狗也罢，但要做一条戏狗，情以何堪。
纵观事态发展，领dao的内心似乎并不愤怒，领dao只是觉得窝囊，那自然，我们也只能跟着觉得窝囊，你哪有上街去表达窝囊的，那岂不是更窝囊。领dao没面子的时候，我们给他们长脸，但领dao有面子的时候，我们被他们掌嘴。我被欺负，我不能游，你被欺负，你让我游，我又情以何堪。你也别说这种民族国土大事应该是我们一起被欺负了，就算政fu不作为，你活的一塌糊涂，也应该挺身而出。我自然可以挺身而出，但我的第一主题就是要求政fu去作为，第二主题才是控诉来犯者，因为领土问题从来都不是老百姓能解决的和该去解决的，尤其是在我国，老百姓自己都没有一寸土地，，所有的一切，都是问政fu租的，所以，理论上，这事对我来说，就是我的房东在和别人就一块在地上的瓦而争执，这块瓦的确是风大的时候从房东的房顶上掉下来的，但房东也不敢去捡，因为可能要和隔壁人家打架。那我等租客在里面搅和什么呢。无土地者要去为他人争取土地，无尊严者要去为他人捍卫尊严，这样的人多少钱一斤？一斤多少个？
但毕竟，这样的you行安全，好玩，显得很酷，关键是游完以后还能正常工作学习，甚至还有助于未来发展，毕竟也算不容易，所以大学生和老百姓抱着尝鲜唱黑脸的角度去游一游无妨。到时候政fu唱一个白脸，说不定能有所见效。况且现在去you行玩的人相比起以前you行玩的人也有着些许不同，以前是彻底的国政不分，被卖数钱，现如今很多青年终于能够将所谓爱国这件事情想的更明白，他们虽然依然愤怒，但开始反思自己为何每次都是那么窝囊和被动，回头也能更客观的看待国家和政fu的关系，这也算是一个进步。对于任何国家来说，国家就是一个女人，zhi zheng者就是占有她的男人，有幸福美满的，有相处和睦的，有家庭暴力的，有关系紧张的，有离婚再嫁的，有不能改嫁的，但无论如何，你爱一个女人总不能连她的男人也一起爱了去。
最后，这些都不重要，最重要的是，我，如果今天能为唐fu珍,谢chao平而you行，那么明天我就一定会为钓 yu岛和奥运火炬而you行。但这又是一个悖论，往往你能够为唐fu珍,谢chao平you行的时候，你往往就不会有钓yu岛奥运火炬之类的事，而且更不会有唐fu珍谢chao平之类的事出现。一个对内不能和平you行的民族，他的对外任何you行是完全没有价值的，那只是一场集体舞。
首先，我认为在现代中国社会中，分为三个阶级，那就是主子，奴才和狗，而我们往往一人饰两角，至于饰演哪两个角色，我想不会有人觉得他在演主子吧。前一阵子，主子需要奴才去附和和伺候，但是现如今，主子需要狗去吼两声，因为在狗的逻辑里，无论主子怎么对待它，只要有外人来犯，狗总是该看家护院的。
当弄明白了这个以后，回头想想就容易多了。但是，在这三个阶级以内，好在我还有选择做花花草草的权力。我的选择依据是，对于相关部门，小事和大事他们的区别就是抗议一次和抗议十一次，有特权有能力的地方尚未出力，除了把人家日本大使变成了应召男郎以外，我们相关部门情绪稳定，并不见什么实际决心，别说武力上，连经济上都不敢有所动作。他们韬光养晦，所以我也韬光养晦。毕竟，我等做狗也罢，但要做一条戏狗，情以何堪。
纵观事态发展，领dao的内心似乎并不愤怒，领dao只是觉得窝囊，那自然，我们也只能跟着觉得窝囊，你哪有上街去表达窝囊的，那岂不是更窝囊。领dao没面子的时候，我们给他们长脸，但领dao有面子的时候，我们被他们掌嘴。我被欺负，我不能游，你被欺负，你让我游，我又情以何堪。你也别说这种民族国土大事应该是我们一起被欺负了，就算政fu不作为，你活的一塌糊涂，也应该挺身而出。我自然可以挺身而出，但我的第一主题就是要求政fu去作为，第二主题才是控诉来犯者，因为领土问题从来都不是老百姓能解决的和该去解决的，尤其是在我国，老百姓自己都没有一寸土地，，所有的一切，都是问政fu租的，所以，理论上，这事对我来说，就是我的房东在和别人就一块在地上的瓦而争执，这块瓦的确是风大的时候从房东的房顶上掉下来的，但房东也不敢去捡，因为可能要和隔壁人家打架。那我等租客在里面搅和什么呢。无土地者要去为他人争取土地，无尊严者要去为他人捍卫尊严，这样的人多少钱一斤？一斤多少个？
但毕竟，这样的you行安全，好玩，显得很酷，关键是游完以后还能正常工作学习，甚至还有助于未来发展，毕竟也算不容易，所以大学生和老百姓抱着尝鲜唱黑脸的角度去游一游无妨。到时候政fu唱一个白脸，说不定能有所见效。况且现在去you行玩的人相比起以前you行玩的人也有着些许不同，以前是彻底的国政不分，被卖数钱，现如今很多青年终于能够将所谓爱国这件事情想的更明白，他们虽然依然愤怒，但开始反思自己为何每次都是那么窝囊和被动，回头也能更客观的看待国家和政fu的关系，这也算是一个进步。对于任何国家来说，国家就是一个女人，zhi zheng者就是占有她的男人，有幸福美满的，有相处和睦的，有家庭暴力的，有关系紧张的，有离婚再嫁的，有不能改嫁的，但无论如何，你爱一个女人总不能连她的男人也一起爱了去。
最后，这些都不重要，最重要的是，我，如果今天能为唐fu珍,谢chao平而you行，那么明天我就一定会为钓 yu岛和奥运火炬而you行。但这又是一个悖论，往往你能够为唐fu珍,谢chao平you行的时候，你往往就不会有钓yu岛奥运火炬之类的事，而且更不会有唐fu珍谢chao平之类的事出现。一个对内不能和平you行的民族，他的对外任何you行是完全没有价值的，那只是一场集体舞。
Sunday, September 12, 2010
韩寒：保住非法字符
有朋友问我，钓鱼岛事件你怎么不发表一点意见，谴责一下日本。我说，虽然脚下一片自己的土地也没有，但对于领土的问题，我也是很在意的。最早的时候，我在一个论坛上看见这事，我义正言辞的写了一句，“保住钓鱼岛”，结果该论坛告诉我，我试图发表非法的内容，请修改。我百思不得其解，直到把帖子改成了“保住尖阁列岛”，这才顺利合法的发表了。
这的确是一件大事，外交部都周末加班破例追加谴责。如果大家一切都好，生活如意，老婆孩子房子车子工作休息健康医疗一切都能保住，闲情雅致之下，民族情操之下，又不愿韬光养晦，当然可以追保钓鱼岛。但是如果你自己还有什么保不住的，先把自己的保住再说，不要操那么前卫的心。也许你会说，在大是大非之下，你个人的小失小患算的了什么，是的，不过每个人都有其自定义大是大非的权力。比如这种事情，我认为先要看政府的态度，你怎么能冲在领导的前面呢？领导表示谴责，意思就是让你表示谴责，领导表示遗憾，意思就是你可以谴责完毕了。领导要谴责，你要动手，这是领导能容忍的极限，你如果真的动手，领导就要惩罚你了，因为领导在下一盘很大的棋子，你身为一个棋子，怎么能跳出棋盘了呢？而在这盘棋子里，你是一颗黑棋，领导就是一颗白棋，一来因为劳动人民总是黑点，而且也容易变成黑户，黑，是你最贴切的颜色，但最关键的是，已经洗白领导要求你在冲锋的时候出来唱黑脸，而领导在关键的时刻唱白脸。事后弄不好你还能发现领导和来犯者还在欢快的谈一笔大生意。
钓鱼岛的问题，我相信我们官方更看重的是自己对内是否稳固，底下石油倒不是那么有所谓，那些都是日本人要的，这也是他们在70年代重新对钓鱼岛起邪念的原因，而中国政府只要稳定，不要在外交和军事上有任何未知的风险，所以这导致了这个本来不复杂的问题一定将被拖延成一个非常复杂的问题。
在我国的版图里，类似有争议有可能引起国家间任何摩擦和局部战乱的地方，只要别太大块，改变了中国是一只鸡的形象，而公众和舆论也不是太了解的犄角旮旯，官方可能觉得让一点也就让一点了，就当卖给地产商了，公鸡母鸡实在是没那么所谓的。钓鱼岛因为一直一来比较有名，公众关注度高，尤其是看了多年新闻联播，领导人都是在钓鱼台国宾馆接见外宾，搞半天钓鱼台都归了别人，这太没面子了，所以，钓鱼岛是政府事关领土问题的形象工程，是底限，我相信这个应该不会让给人家。而对中国政府来说，最好的解决方法就是一直拖着，拖到地壳板块再次发生移动，钓鱼岛直接镶到了福建省，这下就省事了，什么海域石油的事再说。所以我也不担心钓鱼岛被日本人占去，虽然事实上他们的确快占去了。而这次的事件，最好的结果就是船长要被关押十天，在我们强烈谴责严重抗议了九天以后，日本把人放了，我们也算终于抗议有了成果。至于唱黑脸的人们，当然闲来无事唱唱也无妨，只是不要入戏太深，不要影响到自己的生活，不要忘了家人和自己所应该拥有的一切更应该保，不要为发现你已经干了而领导连酒瓶子都没开而伤心，也不要以为自己真的在急民族所最急，这个民族，总有更急的。
http://blog.sina.com.cn/s/blog_4701280b0100lcum.html
这的确是一件大事，外交部都周末加班破例追加谴责。如果大家一切都好，生活如意，老婆孩子房子车子工作休息健康医疗一切都能保住，闲情雅致之下，民族情操之下，又不愿韬光养晦，当然可以追保钓鱼岛。但是如果你自己还有什么保不住的，先把自己的保住再说，不要操那么前卫的心。也许你会说，在大是大非之下，你个人的小失小患算的了什么，是的，不过每个人都有其自定义大是大非的权力。比如这种事情，我认为先要看政府的态度，你怎么能冲在领导的前面呢？领导表示谴责，意思就是让你表示谴责，领导表示遗憾，意思就是你可以谴责完毕了。领导要谴责，你要动手，这是领导能容忍的极限，你如果真的动手，领导就要惩罚你了，因为领导在下一盘很大的棋子，你身为一个棋子，怎么能跳出棋盘了呢？而在这盘棋子里，你是一颗黑棋，领导就是一颗白棋，一来因为劳动人民总是黑点，而且也容易变成黑户，黑，是你最贴切的颜色，但最关键的是，已经洗白领导要求你在冲锋的时候出来唱黑脸，而领导在关键的时刻唱白脸。事后弄不好你还能发现领导和来犯者还在欢快的谈一笔大生意。
钓鱼岛的问题，我相信我们官方更看重的是自己对内是否稳固，底下石油倒不是那么有所谓，那些都是日本人要的，这也是他们在70年代重新对钓鱼岛起邪念的原因，而中国政府只要稳定，不要在外交和军事上有任何未知的风险，所以这导致了这个本来不复杂的问题一定将被拖延成一个非常复杂的问题。
在我国的版图里，类似有争议有可能引起国家间任何摩擦和局部战乱的地方，只要别太大块，改变了中国是一只鸡的形象，而公众和舆论也不是太了解的犄角旮旯，官方可能觉得让一点也就让一点了，就当卖给地产商了，公鸡母鸡实在是没那么所谓的。钓鱼岛因为一直一来比较有名，公众关注度高，尤其是看了多年新闻联播，领导人都是在钓鱼台国宾馆接见外宾，搞半天钓鱼台都归了别人，这太没面子了，所以，钓鱼岛是政府事关领土问题的形象工程，是底限，我相信这个应该不会让给人家。而对中国政府来说，最好的解决方法就是一直拖着，拖到地壳板块再次发生移动，钓鱼岛直接镶到了福建省，这下就省事了，什么海域石油的事再说。所以我也不担心钓鱼岛被日本人占去，虽然事实上他们的确快占去了。而这次的事件，最好的结果就是船长要被关押十天，在我们强烈谴责严重抗议了九天以后，日本把人放了，我们也算终于抗议有了成果。至于唱黑脸的人们，当然闲来无事唱唱也无妨，只是不要入戏太深，不要影响到自己的生活，不要忘了家人和自己所应该拥有的一切更应该保，不要为发现你已经干了而领导连酒瓶子都没开而伤心，也不要以为自己真的在急民族所最急，这个民族，总有更急的。
http://blog.sina.com.cn/s/blog_4701280b0100lcum.html
3 Ways of Making DNA Libraries through Oligo Synthesis
http://allelebiotech.com/blogs/2010/09/3-ways-of-making-dna-libraries-through-oligo-synthesis/
Pools of DNA molecules of related but non-identical sequences are often used for selecting cDNAs that encode polypeptides with desired functions (such as in antibody screening), or DNA segments as protein binding sites (through SELEX), or DNA molecules that can catalyze reactions (DNA enzymes or deoxyribozymes), etc. The most direct way of creating DNA libraries is to introduce mixed bases during the synthesis of the oligos that will be used in creating the libraries.
1) The most commonly used method of generating degenerate oligos is to use mixed phosphoramidites (aka amidites, the building blocks of oligo synthesis) at desired positions in an oligo, e.g. using “N” to incorporate dA, dC, dG, and dT nucleotides, or “Y” for pyrimidines, “R” for purines. Mixed base oligos from most oligo suppliers are simple to order (and at no extra charge from Allele and a few other sources). During automated chemical synthesis of oligos, the synthesizer consecutively adds dT, dA, dC, or dG in the case of “N” at a pre-set ratio (e.g.25% each). This procedure does not always result in expected usage of each amidite because different amidites have different coupling efficiency, and the order of addition may also bias against amidites that are added later.
2) Using mixed bases like in method 1) leaves little control to achieve ratios of codons for specific amino acids. On the other hand, by using trimer amidites, which can be used for adding 3 nucleotides in each synthesis cycle, one can create oligos encoding selected amino acids at pre-determined percentages. However, this procedure is difficult to perform because trimer amidites are bulky and hard to couple to the elongating oligo; any moisture present during synthesis would have even more severe adverse effects than with regular amidites. Trimer oligo synthesis projects cost several thousand dollars per oligo on materials alone, and the risk is quite high that the oligos would not turn out of desired properties and qualities. For commercial users, this process has another problem—it is patented.
3) Another method for making library oligos is the so called “split-and-pool”, which is particularly suitable for having diversified amino acids embedded in otherwise common sequences like the CDRs within antibody variable regions. The latest oligo we made last month was a ~72 nt oligo with 8 locations that have pre-determined composition of amino acids, i.e. 20% Ala, 10% Gly, 12% His, etc. The procedure took us about 8 hours and we estimated the cost to be about $1,000. The subsequent sequencing results confirmed that ~70% of the clones using this oligo have desired degeneracy, compared to a similar oligo made by a bigger oligo company, at only 40%. In addition, we did not see any stop codon interruptions or major abnormalities.
DNA pools can also be generated by error-prone PCR, or more specifically with overlapping PCR using degenerate primers. The bottleneck for a library screening is how to handle big enough a number of colonies to accommodate the population, e.g. 10e10, or at least 10e8 clones are needed for finding high affinity antibodies. The second critical point is to have a robust and consistent selection readout such as fluorescence in cell sorting.
New Product of the Week 090710-091310; loxP-mWasabi reporter T cells, email vivec@allelebiotech.com for details.
Promotion of the Week 090710-091310: 15% off our NEW purified fluorescent proteins (not plasmids); All Expressed from E.coli PROMOCODE: 090910FP
Pools of DNA molecules of related but non-identical sequences are often used for selecting cDNAs that encode polypeptides with desired functions (such as in antibody screening), or DNA segments as protein binding sites (through SELEX), or DNA molecules that can catalyze reactions (DNA enzymes or deoxyribozymes), etc. The most direct way of creating DNA libraries is to introduce mixed bases during the synthesis of the oligos that will be used in creating the libraries.
1) The most commonly used method of generating degenerate oligos is to use mixed phosphoramidites (aka amidites, the building blocks of oligo synthesis) at desired positions in an oligo, e.g. using “N” to incorporate dA, dC, dG, and dT nucleotides, or “Y” for pyrimidines, “R” for purines. Mixed base oligos from most oligo suppliers are simple to order (and at no extra charge from Allele and a few other sources). During automated chemical synthesis of oligos, the synthesizer consecutively adds dT, dA, dC, or dG in the case of “N” at a pre-set ratio (e.g.25% each). This procedure does not always result in expected usage of each amidite because different amidites have different coupling efficiency, and the order of addition may also bias against amidites that are added later.
2) Using mixed bases like in method 1) leaves little control to achieve ratios of codons for specific amino acids. On the other hand, by using trimer amidites, which can be used for adding 3 nucleotides in each synthesis cycle, one can create oligos encoding selected amino acids at pre-determined percentages. However, this procedure is difficult to perform because trimer amidites are bulky and hard to couple to the elongating oligo; any moisture present during synthesis would have even more severe adverse effects than with regular amidites. Trimer oligo synthesis projects cost several thousand dollars per oligo on materials alone, and the risk is quite high that the oligos would not turn out of desired properties and qualities. For commercial users, this process has another problem—it is patented.
3) Another method for making library oligos is the so called “split-and-pool”, which is particularly suitable for having diversified amino acids embedded in otherwise common sequences like the CDRs within antibody variable regions. The latest oligo we made last month was a ~72 nt oligo with 8 locations that have pre-determined composition of amino acids, i.e. 20% Ala, 10% Gly, 12% His, etc. The procedure took us about 8 hours and we estimated the cost to be about $1,000. The subsequent sequencing results confirmed that ~70% of the clones using this oligo have desired degeneracy, compared to a similar oligo made by a bigger oligo company, at only 40%. In addition, we did not see any stop codon interruptions or major abnormalities.
DNA pools can also be generated by error-prone PCR, or more specifically with overlapping PCR using degenerate primers. The bottleneck for a library screening is how to handle big enough a number of colonies to accommodate the population, e.g. 10e10, or at least 10e8 clones are needed for finding high affinity antibodies. The second critical point is to have a robust and consistent selection readout such as fluorescence in cell sorting.
New Product of the Week 090710-091310; loxP-mWasabi reporter T cells, email vivec@allelebiotech.com for details.
Promotion of the Week 090710-091310: 15% off our NEW purified fluorescent proteins (not plasmids); All Expressed from E.coli PROMOCODE: 090910FP
Thursday, September 2, 2010
[转贴]韩寒：草莓
It's been a while since HH published new post, still sharp, still deep, still alive.
http://blog.sina.com.cn/s/blog_4701280b0100l4sf.html
这几天应该是独唱团第二辑上市的时间，由于最近台风登陆，所以还是有所延误，我们在为更好的质量和空间而努力，也为使他变成合法的月刊，而不是居无定所的绝唱团。在这个期间里，GQ杂志为我颁发了一个传媒人奖，这两年，无论是以往的《时尚先生》，还是创刊一年的GQ，都让时尚杂志的男刊不再难堪，很多其他媒体禁忌的名字，甚至出现在他们的名册里，GQ还曾被半路召回，这些都不是业内笑话，而是各种尝试，其实这些都应该是一个传媒人应该去做的。什么是做一个传媒人，我深深的思考过，在我们国家，其实就是做一个传达一下领导意思的媒人，做的不好就送你一个传票，然后就挖煤，这就是传媒人。在很长的一段时间里，我其实是一个被传媒的人，今年，我成了一个所谓的传媒人。好在这个奖项是年度传媒人，它并没有说年度最成功传媒人，我自认为是年度最失败传媒人。失败的原因，以后我会和你们讲来。我们知道，其实很多人宣称的“办一本杂志”“做一份报纸”，都是理想化的称呼，从程序上，这些都是非法的，合法的说法是——某个拥有党委的国有出版社或者杂志社聘请你和你的团队来打理一下他们的杂志。当然，唯一值得欣慰的是，没有我们，他们通常自己的打理的不大好。传媒是一个很大的名词，无论是传统的图书，出版，杂志，报纸，电视，电影，广播，互联网，电子阅读，甚至我们的耳语，打一个电话，贴一张海报，都是传媒，传媒的影响如此之大，谁都想控制它。但传媒其实不该控制在任何人手里，他应该是一片开放的天地，只要善待，谁都可以使用和拥有它，它的上司只有一个，法院，它的罪名只有一种，诽谤。这便是我理想中的传媒。可是理想是每一个人都会说，大家都爱听的，就好比你我都愿面向大海，春暖花开，说一次心里爽一次，却始终无法走出脚下的泥泽。但有念想的人总能走得更远，求生欲强的人总能活的更长，所以，我始终是高兴和乐观的。不过，作为所谓的传媒人，我们总需要不断尝试，我本该很高兴的去北京讲这些话，但因为实在不能过来，所以由老朋友黑狗达代我领一下奖，另外向现场的主编王锋先生，左小诅咒先生，姜文先生，刘北宪先生，白岩松先生和柴静女士问好。向程益中先生问好。
也向刚刚被跨省刑拘的谢朝平先生问好。
这些天一直没有更新这里，我发现在中国写杂文其实是一件很痛苦的事情，虽然这个国家给我们提供了大量的杂文素材，但是你突然发现，写了一阵子以后，当再有新闻出现，你恨不得直接写一句，观点请参考我某年某月写的某一篇。我们的领导群从这一批换成了那一批，治国口号从这一堆换成了那一堆，丰功伟绩从这个会变成了那个会，社会悲剧只是从这个人变成了那个人，换人不换事会让写作者觉得很痛苦，因为大部分的作家都讨厌反复阐述，结果事儿又是反复发生，对我们的遣词造句提出了很大的要求。因为我真的想一直继续写下去，不想让自己厌烦和麻痹。
昨天去看了《盗梦空间》，向大家推荐。
http://blog.sina.com.cn/s/blog_4701280b0100l4sf.html
这几天应该是独唱团第二辑上市的时间，由于最近台风登陆，所以还是有所延误，我们在为更好的质量和空间而努力，也为使他变成合法的月刊，而不是居无定所的绝唱团。在这个期间里，GQ杂志为我颁发了一个传媒人奖，这两年，无论是以往的《时尚先生》，还是创刊一年的GQ，都让时尚杂志的男刊不再难堪，很多其他媒体禁忌的名字，甚至出现在他们的名册里，GQ还曾被半路召回，这些都不是业内笑话，而是各种尝试，其实这些都应该是一个传媒人应该去做的。什么是做一个传媒人，我深深的思考过，在我们国家，其实就是做一个传达一下领导意思的媒人，做的不好就送你一个传票，然后就挖煤，这就是传媒人。在很长的一段时间里，我其实是一个被传媒的人，今年，我成了一个所谓的传媒人。好在这个奖项是年度传媒人，它并没有说年度最成功传媒人，我自认为是年度最失败传媒人。失败的原因，以后我会和你们讲来。我们知道，其实很多人宣称的“办一本杂志”“做一份报纸”，都是理想化的称呼，从程序上，这些都是非法的，合法的说法是——某个拥有党委的国有出版社或者杂志社聘请你和你的团队来打理一下他们的杂志。当然，唯一值得欣慰的是，没有我们，他们通常自己的打理的不大好。传媒是一个很大的名词，无论是传统的图书，出版，杂志，报纸，电视，电影，广播，互联网，电子阅读，甚至我们的耳语，打一个电话，贴一张海报，都是传媒，传媒的影响如此之大，谁都想控制它。但传媒其实不该控制在任何人手里，他应该是一片开放的天地，只要善待，谁都可以使用和拥有它，它的上司只有一个，法院，它的罪名只有一种，诽谤。这便是我理想中的传媒。可是理想是每一个人都会说，大家都爱听的，就好比你我都愿面向大海，春暖花开，说一次心里爽一次，却始终无法走出脚下的泥泽。但有念想的人总能走得更远，求生欲强的人总能活的更长，所以，我始终是高兴和乐观的。不过，作为所谓的传媒人，我们总需要不断尝试，我本该很高兴的去北京讲这些话，但因为实在不能过来，所以由老朋友黑狗达代我领一下奖，另外向现场的主编王锋先生，左小诅咒先生，姜文先生，刘北宪先生，白岩松先生和柴静女士问好。向程益中先生问好。
也向刚刚被跨省刑拘的谢朝平先生问好。
这些天一直没有更新这里，我发现在中国写杂文其实是一件很痛苦的事情，虽然这个国家给我们提供了大量的杂文素材，但是你突然发现，写了一阵子以后，当再有新闻出现，你恨不得直接写一句，观点请参考我某年某月写的某一篇。我们的领导群从这一批换成了那一批，治国口号从这一堆换成了那一堆，丰功伟绩从这个会变成了那个会，社会悲剧只是从这个人变成了那个人，换人不换事会让写作者觉得很痛苦，因为大部分的作家都讨厌反复阐述，结果事儿又是反复发生，对我们的遣词造句提出了很大的要求。因为我真的想一直继续写下去，不想让自己厌烦和麻痹。
昨天去看了《盗梦空间》，向大家推荐。
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