Triple Labeled Oligos-Put Your Fluorescence in Perspective!

Allele is now distributing to its customers the newest technology in RT-PCR published less than two weeks ago. It is a RT PCR probe that utilizes fluorescence resonance energy transfer (FRET) and TaqMan methods in order to provide an internal positive control (IPC) for real-time PCR assays. This new RT-PCR method consists of forward and reverse primers, a regular TaqMan probe, and the new triple-labeled FRET-TaqMan Probe Probe affixed to a plasmid, which negates the uncertainty in the presence and effects of amplification inhibitors that could generate a false negative in your experiments. Two emission sources are all that are required for the FAM and Cy5.5 fluorescence signals at 530 nm and 705 nm respectively. You do not need color compensation software, multiple excitation sources, or even a high tech laser to initiate excitation with this system.

If your RT PCR instrument only has one light source to excite the fluorophore then the FRET-TaqMan method is the only way to exploit the IPC technology. Fortunately Allele Biotech is selling these triple-labeled probes at a great price! Put your fluorescence in perspective and know your true RT-PCR results with these Specialty Oligos from Allele Biotech!

Tags: fluorescence signal, FRET Probe, FRET TaqMan Probe, internal positive control, IPC, led excitation, multiplex real-time PCR, probe, real-time PCR, RT-PCR, special oligonucleotides, TaqMan Probe, tripple-labeled oligos

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

The following information is courtesy of Rick Shindell
at SBIR Gateway, we post this excerpt to here to help more people who may be concerned to become aware of the situation.

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.

Tags: ARRA, GO grant, lab budget, lower costs, NIH challenge grant, NIH funding, research grants, research tools, SBIR

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.

Tags: end fusion, LacZ-based blue/white selection, restriction digesting, TA cloning kit, Taq DNA polymerase, TOPO cloning

Customer Opinions Count!

We are inviting all our customers to give their opinions regarding our products, their experiences with Allele Customer Service, or anything regarding news in biotechnology. Good or bad we want to know what you think!

Tags: Allele service, biotechnology news, customer care, customer opinions, quality service, satisfied customers

Launch of Allele’s Newest Product Group: Special Chemicals

It has only 4 products now, will be a dozen soon, then a few hundreds to thousands in a few months if all goes well. If not, just the oligo chemicals available now should already provide tremendous value to oligo producers because iPCR, Real-time PCR are getting more and more used, requiring large number of probes with these modifications we products can do, but at previously unthinkable low prices. Read about it here.

Tags: amino group, biotin, blocking elongation, capturing oligos, Dabcyl, Dabsyl, FAM, hybridization, iPCR, matching sequences, oligos, probes, real-time PCR