What will be the outcome?

Nobody knows for sure of course, but the number of demonstrators on the street can give Iranian demonstrators unfounded tactical optimism as I felt 20 years ago on Tian'anmen square. No matter how many million people are out on the street, and how determined they are to fight to the end, as the citizens of Beijing were back then, if the government wants to use real force, there will be completely unbalanced unsustainable fight. If it comes to using tanks and machine guns, the only chance will be somehow most of the troops are convinced that shooting or running over unarmed people is an order that can absolutely NOT be carried out.

After seeing Tian'anmen square, military personnel during many mass demonstrations in 1989 Eastern European countries like Romania felt that what they saw on TV a few months ago should not happen in their own country in any case. They'd rather shoot the dictator like in Romania. Maybe reminding everybody of the bloody and horrific street scenes of the Tian'anmen event will be helpful to prevent it from happen again, anywhere.

watching cnn, remembering 20 years ago

This time, from a distance. The show of number by people, the quite period of the power, then the characterization of the movement as against the country, and "western power backing" routine with no details(still no such foreign meddling was eventually shown by evidence 20 years after it was used as one of the reason for cracking down in Tain'anmen square), the escalation on the street.

Iranian people are brave, determined, and very smart. The rooftop shouting at night, the changing of tactics for demonstration, and continuation with communication channels clamped down and clear leaders missing, god I didn't think I'd see history of such scale happen in front of my eyes again in my life. Justice and people's rights will win in the end.

Allele Biotech: From one green revolution to another, Go Freedom in Iran!

The pictures of hundreds of thousands of people on the streets in Tehran eerily reminds me of what I experienced in Tian An Men square: the orderly conducts by the protesters, constant reminding each other to keep lines, maintain non-violent behavior, and obey whatever "orders" that come down from whoever that appeared to be the leaders of the group, as a whole or in small divisions. The crime rates in Beijing before the fateful day of 6.4.89 were at historical lows because citizens never felt that they were so responsible for everything that went on around them. Also similar between the then and now are the blocks on news media, and small incursions between protesters and police forces.

Soon after June 4th, 1989 came the wave of changes in Eastern Europe--people saw what happened in China and they wanted nothing like that in their own country. Hope that is the outcome for Iran.

Allele Will Bring a New Family of Fluorescent Proteins to the Market

Allele has signed an exclusive co-development and marketing agreement with the Swedish high tech company, Innoventus, to work with Dr. Olle Israelsson of the Karolinska Institutet on a novel class of fluorescent proteins.
These proteins were discovered in Amphioxus, a type of small fish that can be found in beach sand, which is believed to be a very primitive cordate species. Compared to jellyfish and coral, from which virtually all of the currently used fluorescent proteins were isolated, Amphoixus are closer to mammalians and their proteins may find great application in human cells and other commonly used animal models. In addition, there are a large number of protein variants that can provide different spectra and other important physical properties such as photostability and photoconvertability.
Allele Biotech’s plan is to first introduce several new fluorescent proteins of different colors to the market as immediate alternatives for fluorescent protein customers. The next step is to continue to evolve and mature these proteins to create more advanced proteins with desired properties suitable for live animal imaging or more advanced applications such as PALM/STORM and SIM. Allele Biotech has on its team of fluorescent protein research staff and collaborators, some of the most highly regarded scientists. With these resources, Allele Biotech plans on committing to long-term development of truly user-friendly fluorescence imaging products.
These new class of fluorescent proteins will be integrated into Allele Biotech’s current products including: retro/lentiviral vectors, baculovirus and bacmam systems, as well as iPSC and RNAi constructs.

Time to renew the SBIR law and the fight is on again.

The following information is courtesy of Rick Shindell
at SBIR Gateway, we are passing it along to make those concerned to become aware.

The four House bills were marked up and approved on June 11, 2009 by the House Small Business Committee's Subcommittee on Contracting and Technology and should go to the full SBC committee next week. The Senate bill is scheduled for markup June 18, 2009.


SENATE SBIR/STTR REAUTHORIZATION BILL S.1233 The Senate's SBIR reauthorization bill was introduced June 10, 2009 and sponsored by SBE committee chair, Mary Landrieu (D-LA), and ranking member Olympia Snowe (R-ME).

At the time of this writing the bill was not yet available from the government printing office, so we can't give you a link to it. We can provide you with an overview. It is close to but not exactly the same as last year.

Important points include:
* Extension of termination dates - 2023 (14 years)
* Improvements to strengthening the SBA Office of Technology
* Increase SBIR allocation by 0.1% per year (starting in FY-2011) until reaching 3.5% in FY-2020
* Increase STTR allocation to .4% for FY-2011; .5% for FY-2013; 0.6% for FY-2015
* Increase SBIR/STTR award levels to $150k phase I and $1M for phase II
* Awards shall not exceed 50% above recommended award levels
* Elimination of Phase II "invitation" process (i.e., DoD)
* VC small biz eligibility compromise limited to 18% of NIH SBIR Award funding, 8% at the other 10 agencies
* Allow small business to partner with federal labs or FFRDC without requiring a wavier from SBA
* Reinstate State and Rural outreach programs
* SBIR STEM Workforce Development Grant Pilot Program
* Continuation of Commercialization Pilot Program (DoD)
* Establish Commercialization Pilot Program for civilian agencies
* Nanotechnology Initiative
* Accelerating Cures - NIH Pilot
* Accuracy In Funding Base Calculations (keep em honest in the 2.5% extramural calculations)
* Increase in technical assistance from $4k to $5k
* SBIR and STTR Special Acquisition Preference

It is highly recommended that if you like the basis of this bill, contact your Senators and ask them to cosponsor this legislation, (S.1233 - A bill to reauthorize and improve the SBIR and STTR programs and for other purposes). This is very important if you want the Senate version to stand a chance on passing.

A tidbit you might have already known, the Challenge Grant through NIH’s ARRA stimulus program received 20k applications for some 200 to 400 awards.

The NIH stimulus grants do not have the SBIR obligations by a last minute change. How may all these affect Allele’s operations? We have submitted 3 grants to the NIH in the last 3 months, with total 4 now pending. It means that we sure are interested in NIH funding, which was, after all, how our company was started. On the other hand, we are also glad that we do have ongoing sales and services that link us directly to users of our technologies. In the current difficult economy and tight funding environment, we strive to be a company that supplies most essential biological research tools that could save average labs some 20-50% cost per item compared to buying from companies like Life Technologies and Clontech, etc. At the same time, we want to provide the convenience to our customers by covering a sufficient number of common reagent areas, a value small specialty companies normally do not offer. See our next blog for more comments on being a flexible and able provider of everything essential.

从大学毕业到毕业十年再到二十年，我思想上最大的变化是什么？（一）

大学毕业后，由于6.4的影响太大、太近、也太突然，我虽然把它当作第一大事向每一个有机会交谈的人谈起，也从没有怀疑过谁是正义一方谁是邪恶一方，但并不能把这一基本的对错观与自己为人处事、安身立命的思想根基联系起来。我的人生的骄傲和支点，或者说我之所以是我和愿意继续是我的原因，还在很大程度上来自于和其他团体、组织、或个人的“privileged” 关联。第一种表现可能就是在向不熟悉自己的人介绍自己的时候会说起自己的大学、中学、甚至小学，不是作为了找寻他乡遇故知的惊喜，而是象动物交手前炸炸毛、亮亮翅以确立自己在每人心目中的地位。如果没提这些则多半儿是不好意思太欺负人，在确认对方“不是个儿”（no match）之后。在听到来往的人大谈“我认识的谁谁谁多么优秀”从而让你感到她/他也如何优秀时，也只是轻微地诧异和有不适感—物以类聚人以群分？至于说到种族、国家背景之类，则是一种脱离政治的潜意识性的反应，即说我国家不好便是在说全国人包括我生活在中国的父老乡亲，这也必然延伸到所有中国种的人或叫华人，包括自己。这种感觉一直延续到毕业后十年左右。

回想起来，这里面还有两个稍微深层的原因。 1）初到美国的不适应感和骤然失去许多以前惯有优势的失落，比如在中国多少年来享受的是北京人、说标准普通话、甚至汉族的正宗、正统的优越感随着初说英文、在美国没有了家庭背景和任何经济支持、变成少数民族等荡然无存。2）受70年代出身论和80年代变种而来的精英论影响，觉得或是希望社会的资源和人们的尊敬应该是按某种已经确立过的结构确定并分配下来。因为我是北大的，所以我已经证明了自己在这一结构中的地位了，没必要请不要打破。记得那时候申请学校的申请表中经常会写“本人来自中国数一数二的大学，它们的录取万里挑一、十万百万里挑一”言下之意凭这个Harvard、Yale都比不上，此时不录取我更待何时。另一个表现就是刚到美国的第一年里凡是自命不凡的命校毕业生的主要精力都花在了转学到“更好”的学校去的热潮中。其结果之一就是反而很多在中国非名校毕业生反而踏踏实实作试验，然后走学术路线开实验室教别人做试验。这里没有对任何职业有任何褒贬的意思。通看全文你就会体会调揩是对所有人和事。

待续

Four ways to clone your PCR products, which one should you use?

Cloning of a PCR fragment is typically achieved by one of the following methods:

1) TOPO cloning using a TOPO blunt vector for PCR products generated with Pfu or many other high fidelity thermostable DNA polymerases, or TOPO TA vector for PCR made with Taq. The efficiency is high and so is the cost partially due to the exclusivity of Life Technologies (Invitrogen) to offer topoisomerase-based cloning vectors. TOPO kits always include competent cells, controls, etc. with the topoisomerase-conjugated vector, further increasing the prices to about $25/reaction. The background of TOPO cloning is normally low as the enzyme-linked ends of the linear vector DNA do not allow self-ligation; however, occasionally circular plasmid formation proceeds through recombining one end of the linear vector to a vector sequence hundreds of bases downstream of the other free end.

2) Restriction digesting PCR fragment for ligating to cloning vector. This method allows you to have the freedom to choose virtually any plasmid vector that immediately suites your experiments intended for using the clone. Unfortunately the efficiency of cutting restriction sites introduced to the ends of linear PCR DNA pieces is often extremely low, even with extra bases added to the outside of the restriction enzyme sites. Background ligation by vector self-ligation or erroneous ligation to contaminated DNA becomes prominent when properly cut insert DNA is not present in sufficient concentration due to poor restriction digestion. The cost should be the lowest using this method, given that you don’t have to do repeated digestion with increasing amount of restriction enzymes and ligation with batch after batch of ligase.

3) End fusion via recombination assisted by end-chewing DNA polymerase or other DNA enzymes. The most actively promoted commercial product for this method is the In-Fusion line from Clontech (Takara). The cost of using the In-Fusion kits is somewhat lower than the TOPO kits, still above $12/reaction in most cases even without accompanying competent cells. At least there is an option of just buying the kit without competent cells, which is basically some dried-down viral DNA polymerase in separate tubes. Because in theory enzyme-assisted recombination can occur between any homologous sequences at the ends of linear DNA, the user has freedom to choose any vector. In practice however, it is questionable how much the added DNA polymerase actually help the efficiency since in our hands we found sometime the no-enzyme controls worked better than using the supplied enzyme. Furthermore, we also observed vector dependence of cloning efficiency.

Preannouncement: Allele Biotech will soon offer a linear form of DNA for both entry point PCR cloning (i.e. putting linear PCR product into a circular vector so that subsequent restriction digestion at the ends of the PCR fragment will be easy) and basic bacterial protein expression. There will be no enzyme required at all and the cost will be much lower than similar products from other suppliers.

4) TA cloning is a simple method of cloning DNA fragments created by a PCR reaction catalyzed by the Taq DNA polymerase. The PCR product with 3’ overhanging A base is ligated to a linear vector with overhanging 5’ T. This method was devised after the discovery that the 3’ ends of PCR products generated by Taq polymerase contain an unmatched A base added by the terminal transferase activity of Taq polymerase. The T/A matching so provided helps increase the ligation efficiency over blunt end ligation such as ligation between Pfu-generated PCR and vectors cut by EcoR V or Sma I. Therefore, even when high fidelity enzymes are used for PCR, many researchers choose to add the extra A base by incubating the PCR product in the presence of Taq for about 15 min. Even though a linear vector with a 5’ overhanging T is not trivial to produce, the cost of TA cloning vectors is still much lower than TOPO or end-fusing vectors. Allele Biotech’s just-launched TA cloning vector also utilizes a LacZ-based blue/white selection. The background ligation rate is very low while ligation with insert typically results in a few hundred colonies by standard procedures using Allele’s Extreme Efficiency competent cells.

Don’t have to remember, because I will never forget

This day 20 years ago was the most dramatic day of our lives so far. The experience has profoundly influenced me to be who I am now and how I view things, even as a private person living a daily life. Maybe I did not see its effect fully then, but I sure feel how it is underpinning everything of substance to me now. Seeing what happened and how it happened taught me in real sense about oppression versus resistance, dishonesty versus being true to self, about what it is so important that a person needs to be fighting till death for, and what is only superficial that one could just let go.

在一场不均衡的战斗中，我只是选择了我所属于的那一方，即使是弱的一方。对曾经站在我身旁却没能一起走出广场的同伴， 我知道我们可能只是随机地交换了地方。我希望我和其他活过来的人以自由和正直的方式度过了我们这些年的多出来的时光，并因此而没让他们失望。