Tips
The procedure for getting a patent
Patent rights are granted by national Patent Offices, and so patent protection for an invention must be sought in each country individually. The procedure generally involves three steps. First, the person (or company) seeking patent protection must file a patent application at the Patent Office.Second, the Patent Office then performs a novelty search, which involves checking all the literature available to it to find documents that describe the invention in part or in whole. In this search, only documents that were published before the date of filing of the application are to be considered.And third, an Examiner decides on the patentability of the invention in light of the report of the novelty search. The applicant of course has the right to discuss the findings of the Examiner, to argue about interpretation of the literature found, and to limit the scope of his patent if necessary. If, after that, there is still an invention left, the applicant is granted a patent for it.
The patent application
The first step in getting a patent on an invention is writing a patent application. In this application, the inventor must disclose the invention in sufficient detail for the average skilled person to be able to reconstruct it. This way, anyone should be able to rebuild the invention and apply it himself once the patent rights run out.
A patent application typically consists of six parts:
- A statement identifying the field of the invention, or the type of apparatus, device, method or other object it relates to.
- An introductory portion, which describes what is known at the time of writing with respect to this field, and which identifies a problem, disadvantage or need that exists therein.
- A brief description of the invention, stating the measures taken in the apparatus or method according to the invention, and the advantages or solutions it brings.
- A detailed description, in the above-mentioned level of detail. This detailed description should mention all aspects of the invention. In some countries it is even required to describe the best way to practice the invention (the "best mode"). In any case, the description should fully describe all aspects of the invention. The detailed description is almost always accompanied by a number of figures. The figures are usually found at the end of the application, except for US patent applications, where they are present at the beginning.
- A number of claims. A claim can be regarded as a definition of what the inventor is trying to claim as "his" invention. There are two types of claims. Independent claims stand on their own; they provide a complete definition. If something else matches the definition of the independent claim, it infringes on the patent. The dependent claims refer back to the independent claims, and provide additional (optional) measures. Should an independent claim be declared invalid, the dependent claims referring back to that claim can be used as a fallback position.Claims can be defined in various categories, such as apparatus, arrangement, device, method, system, computer program, medicine, and so on.
- An abstract, which gives a short description of what the invention is about. The abstract is not legally binding and does not serve to identify the scope in any way - that's what the claims are for. The abstract is there to help readers quickly examine the relevance of the patent.
Writing a patent application is a tricky business, especially when it comes to claim writing. It is common for an inventor to write a detailed description and figures himself, while the claims and the brief description are written by a patent attorney based on the information in the detailed description.
While the claims define the invention, they are to be interpreted in the light of the wording as used in the rest of the application. So, for instance, if a claim uses the word "round", then normally that means exclusively perfectly round. The description, however, might include a phrase like "The shape does not have to be perfectly round", in which case a slight deviation is permissible to still fall under the definition of the claim. The description might even say "The word 'round' is used here to mean any kind of geometrical shape, including but not limited to square, rectangular, elliptical or curved in any other way", in which case almost any shape would be considered to fall under the heading 'round'. Or, as a common saying goes, a patent drafter is his own lexicographer. For more information regarding the scope of a patent and claim interpretation,
The publication
A patent application is published 18 months after it has been filed (or 18 months after its priority application, if any, has been filed). This way, the world is informed about the fact that a patent can be expected on that particular invention. Someone else using the invention could then switch to a different technology, or make reservations to pay for a license once the patent is granted. He could also start looking for relevant prior art (for example, documents on his own use of the invention before the filing date) to get the patent annulled if it is granted eventually.
Until November 2000, the US patent office did not publish applications, but instead only published the granted patents. This meant that someone else using the invention would not have any time to take preventive action. The reason for not publishing is that it robs the inventor from the chance of withdrawing the application if it becomes apparent he will be unable to get a patent on it. Once it has been published, the invention is there for all to see, and the inventor has lost control. If the patent application is subsequently rejected, the inventor can't gain exclusivity through his patent nor through keeping the invention a trade secret
During the examination process, the applicant has the chance to modify his claims, and sometimes part of the description. This means that the patent as granted might differ substantially from the patent application as published. The patent as granted is also published, and the rights granted by a patent becomes effective upon publication.
The novelty search
In order to determine whether an invention is patentable, it must be compared against what was known at the day before the day of filing of the application. This is known as the state of the art. Documents that were not available until on the day of filing are to be disregarded because they were not part of the state of the art at the day of filing
Some countries, most notably the USA, have a so-called "grace period". Publications by the inventor during the grace period, which can range to up to one year before the filing of the patent application, are also not considered to be part of the state of the art. However, this of course only applies to the patent application for the countries in which such a grace period exists. In other countries, the document will be considered of part of the state of the art.
While in general the term "document" it used, in practice any revelation regarding the invention is to be considered to be in the state of the art. So, if the invention was publicly shown at an exhibition before the date of filing, then the presentation is considered to be part of the state of the art. If the inventor told someone about the invention before filing a patent application, then that oral disclosure is part of the state of the art. If such an exhibition or oral disclosure describe all the features of the invention, then the invention is not novel with respect to the state of the art. Proving that the inventor in fact did orally disclose the invention before the patent application was found can be rather difficult, though.
Almost always, the searcher will only consider databases containing printed publications because they are the easiest to search. Based on what he finds in those databases, he draws up a search report which gets published next to the patent application. The applicant can than evaluate the search results and decide whether there is anything patentable left. If so, the patent application moves to the next stage: the examination.
The examination
In this stage, the patent application is compared against the state of the art by an Examiner, who makes a determination whether the invention is novel and whether it involves an inventive step. In order to accurately determine what the invention is, each patent application contains a number of claims at the end of the application. These claims define the invention and indicate for what patent protection is sought. So, the application may describe in very broad wording how the apparatus according to the invention to be constructed, but if the claims described only one specific example, than that specific example is considered to be the invention, regardless of the rest of the application.
To determine whether the invention is novel and involves an inventive step, the Examiner only looks at the claims. If all the elements that are mentioned in the claims are also found in a single document that is part of the state of the art, then the invention is not novel. If the invention is novel, but adding the missing features is considered to be obvious, then the invention lacks an inventive step. The Examiner communicates his findings to the applicant, in the form of a so-called Office Action or Communication.
Generally, the applicant will disagree with the findings of the Examiner. He can then respond to the Communication of the Examiner by pointing out the certain features of the claim are missing in the state of the art, or that a combination of documents made by the Examiner is not proper. He may also introduce additional features in claims, so as to distinguish the invention as claimed from the state of the art.
Often, a claim is followed by a series of so-called dependent claims, which list additional features. Adding a new feature to a claim is then often done by combining the first, independent claim with one or more of the dependent claims. For example, the first claim may describe a method of compressing an image that works by applying run-length encoding on continuous areas of the image. A subsequent dependent claim could then give the additional feature of reducing the number of colors in the color palette. If run-length encoding of continuous areas is found to be present in the state of the art, then the first claim is rejected for lack of novelty. The applicant could then combine the dependent claim and the independent claim to overcome this objection.
The Examiner then has to consider whether a method of compressing an image involving RLE coding and palette adjusting is known in the state of the art. He could then, for example, locate another document which explains that a good compression level is achieved by reducing the number of colors in the palette, and use this document as an argument that the invention lacks an inventive step. The applicant would then have to find a new feature to add to the independent claim or withdraw his application. If the Examiner can find no such document and no such hint anywhere in other documents, then the application apparently does involve an inventive step, and the Examiner will grant a patent.
Of course, a patent is not immune after grant. It is possible to invalidate a patent at any moment based on the fact that it is not novel or that it is obvious over the prior art, as long as the appropriate evidence can be given.
International patent treaties
When someone wants to apply for a patent in multiple countries, he has to follow the national procedures in every one of those countries separately. Since these procedures are roughly the same in all of these countries, this means a lot of duplicated efforts. Various international treaties have been established over the years in order to streamline the application process for a great deal.
The Paris Convention
The oldest treaty related to patents is the Paris Convention (1883). While it does not regulate any aspect of the examination, it does establish the very important right of priority. Someone who has filed a patent application in any country that is a member to the Paris Convention, can within one year after that filing file patent applications in other countries, claiming the filing date of the first application as the effective filing date of the later applications. This way, he has up to one year to decide in which countries he wants to apply for patent protection and to make the necessary preparations (like translating it into the official languages of those countries) for doing so.
When the priority of an earlier application is claimed, the filing date of the earlier application is regarded as the filing date of the later application when doing the novelty search. That means that only publications that exist before the filing date of the earlier application are considered. This allows an inventor to, for example, file a patent application quickly in one country, then publish his invention in a journal or show it at an exhibition, and then at his leisure file patent applications in other countries with the benefit of the priority claim. His publication or presentation at the exhibit will not be damaging for his patent, even though they completely describe the invention and were published before the filing date of those later applications.
An additional advantage of filing an application while claiming the filing date of another application, is that in most countries, the patent term is determined from the actual filing date in that country. This means that this term is effectively shifted one year into the future.
The Patent Cooperation Treaty
Even with the benefit of the priority system as provided by the Paris Convention, obtaining patent protection in 20 countries still means having to start 20 separate national procedures. The Patent Cooperation Treaty (PCT) is an attempt to at least streamline the first two steps of the various national procedures. Using the PCT an applicant needs only to file one single patent application in which he indicates ("designates") all the countries in which he wants to have patent protection.
The International Bureau, a division of the World Intellectual Property Organization (WIPO), receives the patent application and checks that it meets the formal requirements. It also publishes the patent application 18 months after filing. One of the major patent offices in the world is then appointed the International Search Authority and performs the literature search. Uusually this is the US Patent and Trademark Office, the European Patent Office or the Japanese Patent Office. After the search has been performed , and the applicant is satisfied with the results, he can elect to continue the procedure at the National Offices of the countries he designated. The International Bureau transmits the application and the search results to those National Offices. Optionally, the patent office that performed the search can also issue a preliminary opinion on the patentability. This opinion is then also supplied to those National Offices.
Upon receiving a PCT application, the National Offices then immediately start with the examination, often using the search report drawn up by the International Bureau as a basis. This means that the applicant does not need to have a separate search performed at every National Office, which is a substantial saving in costs (although National Offices are free to do their own search from scratch, or perform an supplementary search, which of course still costs money). However, the applicant must still convince each Examiner in every country he designated that the invention is patentable in light of the state of the art as described in the search report. And the Examiner of course applies his country's national standards for patentability.
The European Patent Convention
The European Patent Convention (EPC) goes even beyond what the Patent Cooperation Treaty establishes. An applicant files a single European Patent Application and designates the countries in Europe in which he wants to have patent protection. The European Patent Office (EPO) performs a novelty search and prepares a search report. Using this search report, the Examining Division (three Examiners) then determines the patentability of the invention. The procedure is comparable to the national procedure, except in that it has only to be performed once regardless of how many European countries were designated.
If the Examining Division decides that the invention is patentable, the EPO grants a European Patent. This is a slightly misleading name, since it does not grant any traditional patent rights. Rather, it grants the applicant, in the countries he designated, the same rights as would have been granted in the case of a national application. A European Patent is therefore sometimes referred to as an "bundle of rights".
Despite the name, the EPO is not an organization within the European Union. Also non-EU members are party to the EPC.
Once a European Patent has been granted, anyone has the right to oppose it within 9 months after grant. If the patent is then found to be invalid, it is revoked in all countries simultaneously. After these 9 months, the patent can only be revoked separately for each country in which it was granted. This is substantially more expensive and time-consuming.
The EPO, however, is not the final authority on patent matters in Europe. A European patent effectively grants its owner national patents in every country that is party to the EPO (or those countries the owner designated). Issues of validity and infringement must be dealt with according to the national patent law in each country.
In particular, a European patent can only be declared invalid by a court in one country for that country. Article 16(4) of the Brussels Convention on Jurisdiction and Enforcement specifically states that issues related to the registration of a right are exclusively the domain of the country in which that registration took place. This means that someone wanting to invalidate a European patent that was granted in 18 countries must start 18 separate court proceedings. Of course, once the first few proceedings are won, the rest should be relatively simple and may not even be necessary, since the patent owner will be unlikely to actually enforce his patent after that.
Europe does not have an equivalent of the US Court of Appeals for the Federal Circuit, which means that in principle every country can rule differently on patent matters. There are some restrictions. Article 69 of the EPC gives a basic principle on determination of the scope of the right and the way in which it should be interpreted.
9 Tips on Optimizing Instrument/Equipment Maintenance
Howard Rosenberg, Ph.D.
The life science industry is experiencing watershed changes that are directly driving the need to increase research efficiency while minimizing costs. This “perfect storm” includes pressure to reduce the cost of healthcare, blockbuster drugs reaching their patent end of life, biosimilar proliferation, and weak pipelines—to name a few issues. For individual laboratories, these complex factors often are resulting in ambitious cost saving requirements. One place to look for cost savings is in your equipment and instrument maintenance and repair processes to determine the optimal service levels, service providers, and service types to satisfy those needs most cost-effectively.
1. Repair Price CeilingSet an appropriate equipment replacement price ceiling below which equipment and instruments should be managed on a “run-to-fail” basis and simply replaced upon failure.
2. Criticality Determine the “criticality” (low, medium, high) of each instrument or piece of equipment to your organization. Base the criticality assessment upon a combination of the availability of an alternate instrument/equipment item; availability of an alternate technique to derive the same or similar information; and the impact of the unavailability of the item to your processes and productivity. Never ask a researcher to assess the criticality of a piece of equipment, because the answer will always be that the item is highly critical.
3. Risk of FailureDetermine the risk of failure (low, medium, high) of each piece of equipment or instrument. Risk, in this context, is primarily about the age of item, the maintenance history of the item, and the presence of components that would be very costly to replace if a failure occurred.
4. Service LevelDetermine the appropriate service level for each piece of equipment or instrument according to the criticality and risk assessments. The service level should detail the required response time for corrective maintenance (24 hours, 48 hours, 72 hours) and the type of support:
a. Run to Fail (replace when fail)
b. Run to Fail (repair when fail)
c. Time and Materials (preventative and/or corrective maintenance)
d. Preventative Maintenance Only contract
e. Full Service contract
5. Preventive MaintenanceDetermine the number of preventive maintenance events required for each piece of equipment or instrument. Bear in mind that the original equipment manufacturers’ (OEMs’) recommended number and frequency of preventive maintenance (PM) events are not always appropriate. When determining the proper number of PMs a piece of equipment or instrument should receive, consider such factors as frequency of use, use of fluids in the equipment, number of moving parts, operating environment, and the type of science being conducted.
6. Service ProviderDetermine the appropriate type of service provider for each piece of equipment or instrument. Service provider types include in-house (if available), depot services, third party, and OEM. The type of service provider should be based upon the factors of criticality, risk, and technology level (low, medium, high) of the item. Low-technology equipment includes general lab equipment, freezers, hoods, incubators, centrifuges, etc., while medium-technology equipment includes HPLC, liquid handlers, spectrophotometers, etc. High technology equipment includes spectrometers, analyzers, mass spec, sequencers, etc.
7. Critical PartsDesign and implement a critical parts and “hot swap” program. Critical parts should include PM kits and essential parts for equipment and instruments. Care must be taken in balancing the cost of the parts kept on hand versus the availability and lead time for delivery of critical parts. Some high risk/highly critical equipment such as ultralow temperature freezers used to store irreplaceable samples should be kept ready for immediate use in case of a failure.
8. Computerized Maintenance Management SystemUtilize a computerized maintenance management system (CMMS) to keep track of your laboratory assets, warranty periods, designated service provider, service level agreements, service history, PM schedule, services metrics, etc. While Microsoft Excel can be used to manage some maintenance processes, a true CMMS will manage more information and provide better data and reporting.
9. Equipment Maintenance ProgramPut in place the appropriate resources to manage and run your instrument and equipment maintenance program. Researchers and scientists should never be responsible for the running and managing of the maintenance program as this is not their core function and will consume valuable scientific time, thus reducing their effectiveness. You may want to outsource the management and implementation of your instrument and equipment maintenance program to companies that specialize in this function and free up research staff to focus on their work.
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Howard J. Rosenberg, Ph.D. (Howard.Rosenberg@labwellservices.com), is an svp of Labwell, a division of Jones Lang LaSalle. Ph: (978) 561-1934.
1. Repair Price CeilingSet an appropriate equipment replacement price ceiling below which equipment and instruments should be managed on a “run-to-fail” basis and simply replaced upon failure.
2. Criticality Determine the “criticality” (low, medium, high) of each instrument or piece of equipment to your organization. Base the criticality assessment upon a combination of the availability of an alternate instrument/equipment item; availability of an alternate technique to derive the same or similar information; and the impact of the unavailability of the item to your processes and productivity. Never ask a researcher to assess the criticality of a piece of equipment, because the answer will always be that the item is highly critical.
3. Risk of FailureDetermine the risk of failure (low, medium, high) of each piece of equipment or instrument. Risk, in this context, is primarily about the age of item, the maintenance history of the item, and the presence of components that would be very costly to replace if a failure occurred.
4. Service LevelDetermine the appropriate service level for each piece of equipment or instrument according to the criticality and risk assessments. The service level should detail the required response time for corrective maintenance (24 hours, 48 hours, 72 hours) and the type of support:
a. Run to Fail (replace when fail)
b. Run to Fail (repair when fail)
c. Time and Materials (preventative and/or corrective maintenance)
d. Preventative Maintenance Only contract
e. Full Service contract
5. Preventive MaintenanceDetermine the number of preventive maintenance events required for each piece of equipment or instrument. Bear in mind that the original equipment manufacturers’ (OEMs’) recommended number and frequency of preventive maintenance (PM) events are not always appropriate. When determining the proper number of PMs a piece of equipment or instrument should receive, consider such factors as frequency of use, use of fluids in the equipment, number of moving parts, operating environment, and the type of science being conducted.
6. Service ProviderDetermine the appropriate type of service provider for each piece of equipment or instrument. Service provider types include in-house (if available), depot services, third party, and OEM. The type of service provider should be based upon the factors of criticality, risk, and technology level (low, medium, high) of the item. Low-technology equipment includes general lab equipment, freezers, hoods, incubators, centrifuges, etc., while medium-technology equipment includes HPLC, liquid handlers, spectrophotometers, etc. High technology equipment includes spectrometers, analyzers, mass spec, sequencers, etc.
7. Critical PartsDesign and implement a critical parts and “hot swap” program. Critical parts should include PM kits and essential parts for equipment and instruments. Care must be taken in balancing the cost of the parts kept on hand versus the availability and lead time for delivery of critical parts. Some high risk/highly critical equipment such as ultralow temperature freezers used to store irreplaceable samples should be kept ready for immediate use in case of a failure.
8. Computerized Maintenance Management SystemUtilize a computerized maintenance management system (CMMS) to keep track of your laboratory assets, warranty periods, designated service provider, service level agreements, service history, PM schedule, services metrics, etc. While Microsoft Excel can be used to manage some maintenance processes, a true CMMS will manage more information and provide better data and reporting.
9. Equipment Maintenance ProgramPut in place the appropriate resources to manage and run your instrument and equipment maintenance program. Researchers and scientists should never be responsible for the running and managing of the maintenance program as this is not their core function and will consume valuable scientific time, thus reducing their effectiveness. You may want to outsource the management and implementation of your instrument and equipment maintenance program to companies that specialize in this function and free up research staff to focus on their work.
--
Howard J. Rosenberg, Ph.D. (Howard.Rosenberg@labwellservices.com), is an svp of Labwell, a division of Jones Lang LaSalle. Ph: (978) 561-1934.
10 Tips to Improve Pipetting Technique
George Rodrigues, Ph.D.
Of all the factors contributing to performance, the most critical are skill and expertise of the operator.
1. Prewet the pipette tipAspirate and fully expel an amount of the liquid at least three times before aspirating for delivery. Failure to prewet increases evaporation within the tip air space, which can cause significantly lower delivery volumes. Prewetting increases the humidity within the tip, thus reducing evaporation.
2. Work at temperature equilibriumAllow liquids and equipment to equilibrate to ambient temperature prior to pipetting. The volume of liquid delivered by air displacement pipettes varies with relative humidity and vapor pressure of the liquid—both of which are temperature-dependent. Working at a constant temperature minimizes variation of pipetted volume.
3. Examine the tip before and after dispensing sampleBefore dispensing, carefully remove droplets on the outside of the tip with a lint-free cloth, being sure to stay clear of the tip opening to avoid wicking liquid out of the tip. After dispensing and before releasing the plunger, deliver any residual liquid remaining in the tip by touching the tip to the side of the container. Surface tension will help draw the remaining liquid out of the tip.
4. Use standard mode pipettingDepress the plunger to the first stop, immerse the tip into the liquid, and aspirate by releasing the plunger. Remove the pipette from the liquid, and depress the plunger to the second stop to dispense the entire contents. Standard (or forward) mode pipetting yields better accuracy and precision than reverse mode for all but viscous or volatile liquids. Reverse mode often results in over-delivery.
5. Pause consistently after aspirationAfter aspirating and before removing the tip from the liquid, pause for one second. Make this pause as consistent as possible Liquid continues to flow into the tip for a short time after the plunger stops. At the same time, evaporation within the tip is occurring. Pausing consistently balances these two effects and ensures correct aspiration.
6. Pull the pipette straight outWhen aspirating, hold the pipette vertically and pull the pipette straight out from the center of the container. This technique is especially important when pipetting small volumes (less than 50 µL). Holding the pipette at an angle as it is removed from the liquid alters the volume aspirated. Touching the sides of the container causes wicking and loss of volume.
7. Minimize handling of the pipette and tipHold the pipette loosely, return it to the pipette stand or set it down between deliveries. Avoid handling pipette tips or containers of liquid to be pipetted. Body heat transferred during handling disturbs temperature equilibrium, which leads to variations in delivered volume.
8. Immerse the tip to the proper depthBefore aspirating, immerse the tip adequately below the meniscus. Large volume pipettes (1–5 mL) should be immersed 5–6 mm, while smaller volume pipettes should be immersed 2–3 mm.
Too little immersion, particularly with large volume pipettes, can lead to aspiration of air. Too much immersion can cause liquid to cling to the outside of the tip. Contacting the container bottom with the tip may restrict aspiration.
9. Use the correct pipette tipUse high-quality tips intended for use with the pipette. In most cases, manufacturer tips perform well. Alternate brands are also acceptable if their performance has been proven with the pipette model. Mismatched tips and pipettes can result in inaccuracy, imprecision, or both. Quality tips provide an airtight seal, are made of superior materials, and are free of molding defects—thereby ensuring dependable liquid delivery.
10. Use consistent plunger pressure and speedDepress the plunger smoothly until coming to rest with a light and consistent force at the first stop. Immerse the tip, then release the plunger at a constant rate. It’s all about rhythm—repeatable actions produce repeatable results.
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George Rodrigues, Ph.D., is senior scientific manager, Artel, a leader in liquid-handling quality assurance. The company holds regular webinars and training sessions to teach the best practices in manual pipetting. For more information, please visit www.artel-usa.com/learning_center.aspx.
1. Prewet the pipette tipAspirate and fully expel an amount of the liquid at least three times before aspirating for delivery. Failure to prewet increases evaporation within the tip air space, which can cause significantly lower delivery volumes. Prewetting increases the humidity within the tip, thus reducing evaporation.
2. Work at temperature equilibriumAllow liquids and equipment to equilibrate to ambient temperature prior to pipetting. The volume of liquid delivered by air displacement pipettes varies with relative humidity and vapor pressure of the liquid—both of which are temperature-dependent. Working at a constant temperature minimizes variation of pipetted volume.
3. Examine the tip before and after dispensing sampleBefore dispensing, carefully remove droplets on the outside of the tip with a lint-free cloth, being sure to stay clear of the tip opening to avoid wicking liquid out of the tip. After dispensing and before releasing the plunger, deliver any residual liquid remaining in the tip by touching the tip to the side of the container. Surface tension will help draw the remaining liquid out of the tip.
4. Use standard mode pipettingDepress the plunger to the first stop, immerse the tip into the liquid, and aspirate by releasing the plunger. Remove the pipette from the liquid, and depress the plunger to the second stop to dispense the entire contents. Standard (or forward) mode pipetting yields better accuracy and precision than reverse mode for all but viscous or volatile liquids. Reverse mode often results in over-delivery.
5. Pause consistently after aspirationAfter aspirating and before removing the tip from the liquid, pause for one second. Make this pause as consistent as possible Liquid continues to flow into the tip for a short time after the plunger stops. At the same time, evaporation within the tip is occurring. Pausing consistently balances these two effects and ensures correct aspiration.
6. Pull the pipette straight outWhen aspirating, hold the pipette vertically and pull the pipette straight out from the center of the container. This technique is especially important when pipetting small volumes (less than 50 µL). Holding the pipette at an angle as it is removed from the liquid alters the volume aspirated. Touching the sides of the container causes wicking and loss of volume.
7. Minimize handling of the pipette and tipHold the pipette loosely, return it to the pipette stand or set it down between deliveries. Avoid handling pipette tips or containers of liquid to be pipetted. Body heat transferred during handling disturbs temperature equilibrium, which leads to variations in delivered volume.
8. Immerse the tip to the proper depthBefore aspirating, immerse the tip adequately below the meniscus. Large volume pipettes (1–5 mL) should be immersed 5–6 mm, while smaller volume pipettes should be immersed 2–3 mm.
Too little immersion, particularly with large volume pipettes, can lead to aspiration of air. Too much immersion can cause liquid to cling to the outside of the tip. Contacting the container bottom with the tip may restrict aspiration.
9. Use the correct pipette tipUse high-quality tips intended for use with the pipette. In most cases, manufacturer tips perform well. Alternate brands are also acceptable if their performance has been proven with the pipette model. Mismatched tips and pipettes can result in inaccuracy, imprecision, or both. Quality tips provide an airtight seal, are made of superior materials, and are free of molding defects—thereby ensuring dependable liquid delivery.
10. Use consistent plunger pressure and speedDepress the plunger smoothly until coming to rest with a light and consistent force at the first stop. Immerse the tip, then release the plunger at a constant rate. It’s all about rhythm—repeatable actions produce repeatable results.
--
George Rodrigues, Ph.D., is senior scientific manager, Artel, a leader in liquid-handling quality assurance. The company holds regular webinars and training sessions to teach the best practices in manual pipetting. For more information, please visit www.artel-usa.com/learning_center.aspx.
7 Tips to Make PCR Primers Last Longer and PCR Reactions Run Better
Tom Russell, Ph.D., Eric Reyes
Successful PCR runs require more than excellent primer design. Here are seven best practices for both obtaining consistently high-quality data and ensuring the integrity and economic use of your PCR reagents.
1. Centrifuge lyophilized primers upon delivery. While the DNA is usually present as a nearly invisible film on the bottom of the tube, it can come loose and fall out when the cap is removed for the first time.
2. Resuspend primers in 10 mM Tris pH 7.5, 1 mM EDTA solution (TE buffer) instead of water. The Tris and EDTA prevent acidic water and contaminating DNases from hydrolyzing and enzymatically degrading DNA, respectively.
3. Aliquot the resuspended primers into working stocks. This eliminates the need for damaging freeze / thaw cycles of the master stock as the working stocks can be removed from the freezer three to five times without degrading the DNA. If contamination of a working stock occurs, it can be thrown out and replaced with another without compromising the master stock.
4. As with your primers, if you purchase large volumes (e.g., 50 mL) of 2X PCR premixes, you can aliquot these reagents into smaller working stocks that are suitable for a single experiment (e.g., a 96-well plate) to avoid contamination.
5. For each target gene you are working on, make a “master mix” of your 2X PCR premix, water, and primers. Some people factor in extra replicates or a percentage (~10%) of volume for each master mix to account for pipetting error. You can then add this “master mix” to the tubes/plates first and then add your samples.
6. If you want to be “extra-passionate” about the precision between your technical replicates, you can even make a “master mix” for each target and sample—add the 2X PCR premix, primers, water, and template—and then add everything all at once into your tubes/plates. This will require extra tubes, but the intra-replicate variability can be greatly reduced.
7. Always keep your pipettes calibrated and take your time. Reagents are expensive, and samples can be limited.
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Tom Russell, Ph.D., and Eric Reyes are global product managers for oligonucleotides and PCR Reagents, respectively, at Sigma Life Science. To learn more about Sigma Life Science’s PCR reagents and online oligo design tools, click here.
1. Centrifuge lyophilized primers upon delivery. While the DNA is usually present as a nearly invisible film on the bottom of the tube, it can come loose and fall out when the cap is removed for the first time.
2. Resuspend primers in 10 mM Tris pH 7.5, 1 mM EDTA solution (TE buffer) instead of water. The Tris and EDTA prevent acidic water and contaminating DNases from hydrolyzing and enzymatically degrading DNA, respectively.
3. Aliquot the resuspended primers into working stocks. This eliminates the need for damaging freeze / thaw cycles of the master stock as the working stocks can be removed from the freezer three to five times without degrading the DNA. If contamination of a working stock occurs, it can be thrown out and replaced with another without compromising the master stock.
4. As with your primers, if you purchase large volumes (e.g., 50 mL) of 2X PCR premixes, you can aliquot these reagents into smaller working stocks that are suitable for a single experiment (e.g., a 96-well plate) to avoid contamination.
5. For each target gene you are working on, make a “master mix” of your 2X PCR premix, water, and primers. Some people factor in extra replicates or a percentage (~10%) of volume for each master mix to account for pipetting error. You can then add this “master mix” to the tubes/plates first and then add your samples.
6. If you want to be “extra-passionate” about the precision between your technical replicates, you can even make a “master mix” for each target and sample—add the 2X PCR premix, primers, water, and template—and then add everything all at once into your tubes/plates. This will require extra tubes, but the intra-replicate variability can be greatly reduced.
7. Always keep your pipettes calibrated and take your time. Reagents are expensive, and samples can be limited.
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Tom Russell, Ph.D., and Eric Reyes are global product managers for oligonucleotides and PCR Reagents, respectively, at Sigma Life Science. To learn more about Sigma Life Science’s PCR reagents and online oligo design tools, click here.
8 Tips for Increasing Upstream and Downstream Productivity and Efficiency
Jeffery Lee Craig
The continuing need to increase titers while decreasing capacity, combined with the resurgence in vaccines and the introduction of gene and cell therapies, mean that efficiency and productivity are the key elements in upstream biopharmaceutical manufacture. As these increased titers move downstream, the increased processing flexibility and reduction in the number of purifications steps afforded by single-use technology, can often be utilized to optimize manufacturing operations. Here are steps to increase productivity as well as efficiency upstream and downstream.
1. Seek to decrease downstream bottlenecks by increasing upstream productivity. Remain focused on process intensification, optimization, and expansion.
2. Upstream process intensification can be achieved with higher titers in smaller bioreactor volumes.
3. Upstream process optimization can be achieved with higher titers by optimizing gas sparging, low shear mixing, and higher cell viability and density.
4. Upstream process expansion can be achieved by replicating, at a larger scale, exact microenvironment conditions and consolidating multiple manual steps in an automated bioreactor with larger surface.
5. Seek to increase downstream efficiency through the use of best in class single-use technology.
6. Maximize vessel integrity through use of fully assembled bags and manifolds that are 100% integrity tested by pressure decay methods after assembly and before irradiation.
7. Maximize vessel integrity by use of fully assembled bags and manifolds through Helium Integrity Testing (HIT) for pinholes as small as 10 microns after assembly and before irradiation.
8. Use mixers that do not shed particles or grind contents.
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Jeffery Lee Craig is global director of business development and marketing at ATMI LifeSciences.
1. Seek to decrease downstream bottlenecks by increasing upstream productivity. Remain focused on process intensification, optimization, and expansion.
2. Upstream process intensification can be achieved with higher titers in smaller bioreactor volumes.
3. Upstream process optimization can be achieved with higher titers by optimizing gas sparging, low shear mixing, and higher cell viability and density.
4. Upstream process expansion can be achieved by replicating, at a larger scale, exact microenvironment conditions and consolidating multiple manual steps in an automated bioreactor with larger surface.
5. Seek to increase downstream efficiency through the use of best in class single-use technology.
6. Maximize vessel integrity through use of fully assembled bags and manifolds that are 100% integrity tested by pressure decay methods after assembly and before irradiation.
7. Maximize vessel integrity by use of fully assembled bags and manifolds through Helium Integrity Testing (HIT) for pinholes as small as 10 microns after assembly and before irradiation.
8. Use mixers that do not shed particles or grind contents.
--
Jeffery Lee Craig is global director of business development and marketing at ATMI LifeSciences.
12 Tips for Fluorescent Multiplex Blotting
Paul Liu, Ph.D.
Anxious when it comes to performing fluorescent multiplex western blotting? Don’t worry, we have 12 tips that will make you a pro:
1. Use primary antibodies from different host species (for example, mouse and rabbit). Antibodies produced from two closely related species such rat and mouse often give cross-reactivity, even when the antibodies are cross-adsorbed.
2. Use secondary antibodies that are highly cross-adsorbed against other species to avoid cross-reactivity.
3. Always optimize the detection of each target singly before simultaneous detection of multiple targets. As some primary antibodies may be nonspecific and yield multiple bands on a blot, a single target detection will help determine the banding pattern of each antibody prior to a multiplex experiment. Antibody concentrations should be optimized by incubating the membrane in several dilutions of each antibody. Select the dilution that yields the highest signal to background ratio.
4. Detect the strongest target in the blue channel, the middle target in the green channel, and reserve the red channel for the weakest target. Most membranes show higher background with shorter wavelength excitation light.
5. Use fluorophores conjugated to secondary antibodies with distinct spectra so they can be optically distinguished from each other to avoid cross-channel fluorescence (i.e., red, green, and blue).
6. When adapting a chemiluminescent protocol to fluorescent detection, primary antibody concentrations may need to be increased two- to five-fold. Secondary antibody concentrations may also have to be optimized; a good starting point is a 1:5,000 dilution.
7. In order to maximize the signal to background ratio, use a membrane with low auto-fluorescence, such as a low fluorescence PVDF membrane.
8. For blocking buffer, use 0.5–5% casein and up to 5% nonfat dry milk or up to 3% BSA dissolved in TTBS. Particulates in the buffer can settle on membranes and create fluorescent artifacts; use only high-quality reagents and/or filter sterilize all buffers.
9. Use blunt forceps to handle the membrane from the edge and avoid scratching or creasing the membrane, which can produce artifacts during fluorescent detection.
10. Use pencil to mark membranes as many inks fluoresce.
11. To avoid fluorescent bromophenol blue from interfering with your gel images, do one of the following: 1) ensure the dye front has migrated away from all samples, 2) cut off the portion of the gel containing the dye front, or 3) omit bromophenol blue from the sample buffer.
12. It is not necessary to perform immunodetection in the dark as normal lighting will not significantly photobleach fluorescently labeled antibodies. However, store stocks of fluorescently labeled antibodies in the dark.
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Paul Z. Liu, Ph.D., is a senior scientist at Bio-Rad, a leading provider of Western blotting equipment. For more information please visit bio-rad.com/ad/V3.
1. Use primary antibodies from different host species (for example, mouse and rabbit). Antibodies produced from two closely related species such rat and mouse often give cross-reactivity, even when the antibodies are cross-adsorbed.
2. Use secondary antibodies that are highly cross-adsorbed against other species to avoid cross-reactivity.
3. Always optimize the detection of each target singly before simultaneous detection of multiple targets. As some primary antibodies may be nonspecific and yield multiple bands on a blot, a single target detection will help determine the banding pattern of each antibody prior to a multiplex experiment. Antibody concentrations should be optimized by incubating the membrane in several dilutions of each antibody. Select the dilution that yields the highest signal to background ratio.
4. Detect the strongest target in the blue channel, the middle target in the green channel, and reserve the red channel for the weakest target. Most membranes show higher background with shorter wavelength excitation light.
5. Use fluorophores conjugated to secondary antibodies with distinct spectra so they can be optically distinguished from each other to avoid cross-channel fluorescence (i.e., red, green, and blue).
6. When adapting a chemiluminescent protocol to fluorescent detection, primary antibody concentrations may need to be increased two- to five-fold. Secondary antibody concentrations may also have to be optimized; a good starting point is a 1:5,000 dilution.
7. In order to maximize the signal to background ratio, use a membrane with low auto-fluorescence, such as a low fluorescence PVDF membrane.
8. For blocking buffer, use 0.5–5% casein and up to 5% nonfat dry milk or up to 3% BSA dissolved in TTBS. Particulates in the buffer can settle on membranes and create fluorescent artifacts; use only high-quality reagents and/or filter sterilize all buffers.
9. Use blunt forceps to handle the membrane from the edge and avoid scratching or creasing the membrane, which can produce artifacts during fluorescent detection.
10. Use pencil to mark membranes as many inks fluoresce.
11. To avoid fluorescent bromophenol blue from interfering with your gel images, do one of the following: 1) ensure the dye front has migrated away from all samples, 2) cut off the portion of the gel containing the dye front, or 3) omit bromophenol blue from the sample buffer.
12. It is not necessary to perform immunodetection in the dark as normal lighting will not significantly photobleach fluorescently labeled antibodies. However, store stocks of fluorescently labeled antibodies in the dark.
--
Paul Z. Liu, Ph.D., is a senior scientist at Bio-Rad, a leading provider of Western blotting equipment. For more information please visit bio-rad.com/ad/V3.
5 Tips to Improve Your RNAi Screens
Andrea Spencer
RNAi is a powerful tool for elucidating gene function, especially when conducted at a whole-genome scale and in a quantitative context. Following these tips can ensure an RNAi screen can be conducted quickly, economically, and in a manner that the data can be readily interpreted.
1. Although lentiviral particles produced using the appropriate packaging plasmids are replication incompetent, it is recommended that they be treated as Risc Group Level 2 (RGL-2) organisms for laboratory handling. Also, use extra caution with lentiviral particles that express shRNAs targeting both cell cycle control and tumor suppressor genes.
2. Determining the viral titer is critical to ensure consistent transduction conditions within a screen and from one experiment to the next. An indirect titering procedure such as measuring p24 viral protein is reliable only if an optimized and standardized viral production procedure is followed. To fully understand how well the lentivirus will transduce a cell line of interest, one must conduct a functional titering assay such as the colony forming unit (cfu) assay.
3. Cells transduced with an “empty” (no shRNA construct) or a nontargeting shRNA should be included in all experiments as controls. Such controls help account for any changes in gene expression that might occur due to passaging, treatments, and selection.
4. When conducting screens, using a redundant shRNA library, which contains multiple shRNA sequences targeting the same mRNA, helps mitigate the potential complication of off-target effects.
5. When conducting pooled-shRNA screens, it is ideal to set up transductions at an MOI < 1 to ensure that cells are transduced with a single shRNA-encoding viral particle. This allows for the shRNA responsible for a phenotype of interest to be identified during downstream deconvolution.
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Andrea Spencer is a senior scientist at Sigma Life Science. To learn more about gene silencing reagents (for siRNA, esiRNA, shRNA, and miRNA), click here.
1. Although lentiviral particles produced using the appropriate packaging plasmids are replication incompetent, it is recommended that they be treated as Risc Group Level 2 (RGL-2) organisms for laboratory handling. Also, use extra caution with lentiviral particles that express shRNAs targeting both cell cycle control and tumor suppressor genes.
2. Determining the viral titer is critical to ensure consistent transduction conditions within a screen and from one experiment to the next. An indirect titering procedure such as measuring p24 viral protein is reliable only if an optimized and standardized viral production procedure is followed. To fully understand how well the lentivirus will transduce a cell line of interest, one must conduct a functional titering assay such as the colony forming unit (cfu) assay.
3. Cells transduced with an “empty” (no shRNA construct) or a nontargeting shRNA should be included in all experiments as controls. Such controls help account for any changes in gene expression that might occur due to passaging, treatments, and selection.
4. When conducting screens, using a redundant shRNA library, which contains multiple shRNA sequences targeting the same mRNA, helps mitigate the potential complication of off-target effects.
5. When conducting pooled-shRNA screens, it is ideal to set up transductions at an MOI < 1 to ensure that cells are transduced with a single shRNA-encoding viral particle. This allows for the shRNA responsible for a phenotype of interest to be identified during downstream deconvolution.
--
Andrea Spencer is a senior scientist at Sigma Life Science. To learn more about gene silencing reagents (for siRNA, esiRNA, shRNA, and miRNA), click here.
10 Tips for Translational Stem Cell Therapy Research
Shawna M. Jackman, Ph.D.
Preclinical studies of stem cell therapies are critical for FDA approval to begin clinical trials, but there currently are no guidelines that address this important area of preclinical research. Here are some tips to consider when designing translational cell therapy research:
1. Ensure each step in the program is customized to the particular cell line, its actions, and the intended clinical use.
2. Understand the cell product. Cell products must be well-characterized with established production processes, detection methods, and characterization specifications before the initiation of preclinical studies and must be the same as the product to be used in clinical trials.
3. Animal models must be both clinically relevant and appropriate for preclinical study objectives.
4. Animal model selection must consider biological relevance, activity, persistence, and migration/distribution, and all must be relevant to clinical use.
5. Specialized animal models may be required (e.g., animal models for the targeted disease or the use of a specific immunocompromised model).
6. Consider limitations of administering the clinically relevant dose and immunogenicity concerns.
7. Ensure that the method of delivery does not confound toxicity.
8. Develop, optimize, and validate translational biomarkers (e. g., morphology, surface, and/or genetic marker) and the methods for cell detection (e.g., PCR, IHC) during pilot studies. These methods must be robust, reliable, and sensitive.
9. Thoroughly evaluate cell phenotypic stability in vivo to clarify the potential for tumorigenicity and/or ectopic tissue formation.
10. Communicate with regulatory agencies on preclinical program designs and animal model selections before studies are initiated.
Strategic program planning and design for these unique cellular therapies lead to successful preclinical programs and ultimately, successful therapies.
--
Shawna M. Jackman, Ph.D., Senior Research Scientist at Charles River. To learn more about Charles River science,
please visit Eureka.
1. Ensure each step in the program is customized to the particular cell line, its actions, and the intended clinical use.
2. Understand the cell product. Cell products must be well-characterized with established production processes, detection methods, and characterization specifications before the initiation of preclinical studies and must be the same as the product to be used in clinical trials.
3. Animal models must be both clinically relevant and appropriate for preclinical study objectives.
4. Animal model selection must consider biological relevance, activity, persistence, and migration/distribution, and all must be relevant to clinical use.
5. Specialized animal models may be required (e.g., animal models for the targeted disease or the use of a specific immunocompromised model).
6. Consider limitations of administering the clinically relevant dose and immunogenicity concerns.
7. Ensure that the method of delivery does not confound toxicity.
8. Develop, optimize, and validate translational biomarkers (e. g., morphology, surface, and/or genetic marker) and the methods for cell detection (e.g., PCR, IHC) during pilot studies. These methods must be robust, reliable, and sensitive.
9. Thoroughly evaluate cell phenotypic stability in vivo to clarify the potential for tumorigenicity and/or ectopic tissue formation.
10. Communicate with regulatory agencies on preclinical program designs and animal model selections before studies are initiated.
Strategic program planning and design for these unique cellular therapies lead to successful preclinical programs and ultimately, successful therapies.
--
Shawna M. Jackman, Ph.D., Senior Research Scientist at Charles River. To learn more about Charles River science,
please visit Eureka.
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