Frequently Asked Questions
The FAQ below is built from selected questions from the web site of Applied Biosystems, the equipment manufacturer and supplier for the Real-Time PCR Core. The questions included here represent those most frequently encountered in the course of operations at our core. The entire Applied Biosystems FAQ may be found here
- Can I use my MGB probe for a Real Time TaqMan® run?
- It seems like I usually don't get a protocol shipped with my Applied Biosystems reagents. Why not?
- Is there a User Manual for Primer Express® that I can download from the Applied Biosystems web page?
- I'm not sure what dye was used to label the probe in the Pre-Developed TaqMan® Assay reagents (PDAR) that I'm using. How can I find out?
- Why am I able to read wells containing the VIC™ dye when I am looking at the FAM dye layer?
- Is it possible to run a multiplex RT-PCR on the ABI PRISM® 7000 or ABI Prism® 7900HT Sequence Detection System, and can I use more than two dyes in a single tube?
- What is the advantage of using VIC™ dye instead of JOE™ or HEX dye for a TaqMan® probe?
- Can AmpliTaq Gold™ be used in a one-step or a two-step RT-PCR?
- Does AmpliTaq Gold™ leave an A overhang as does AmpliTaq® DNA Polymerase?
- Does the activation of AmpliTaq Gold™ at 95°C for 10 minutes interfere with the half-life of the enzyme?
- Once AmpliTaq Gold™ is activated does it remain in an active state?
- Why does amplification efficiency decrease or plateau in the late cycles?
- What is "Hot-start" PCR?
- Does Applied Biosystems have a document that contains chemistry optimization guidelines for the ABI PRISM® 7000, ABI PRISM® 7900HT?
- Can Relative Quantitation of Gene Expression be performed on the ABI PRISM® 7700 Sequence Detection System?
- What can be used to dilute my TaqMan® probe?
- Does AmpliTaq Gold® DNA Polymerase contain 3'- 5' exonuclease (proofreading) activity?
- How does AmpliTaq Gold® DNA Polymerase differ from AmpliTaq® DNA Polymerase?
- How do I use Primer Express for designing primer and probe sets for TaqMan®?
- Where can I find information about TaqMan® Assays and the ABI Prism® Sequence Detection Systems?
- Can I use Oligo (dT)16 for my RT reactions in conjunction with the TaqMan® Human Endogenous control Plate (P/N 4309199)?
- How can I download a SDS protocol?
- What is the minimum/maximum reaction volume I should be using in the ABI PRISM® 7000 and ABI Prism® 7900HT?
- Can I collect my data on the PC connected to the ABI Prism® 7000/7900HT and analyze it on another PC?
- What does the Core provide?
Q1. Can I use my MGB probe for a Real Time TaqMan® run?
Yes, you can use a MGB probe for Real Time PCR, but only when using SDS v.1.7 analysis software. Make sure that you change the quencher to NONE by going to Sample Type: Sample Type Setup on your plate, and selecting NONE.
Q2. It seems like I usually don't get a protocol shipped with my Applied Biosystems reagents. Why not?
Because we have so many repeat customers, we do not send a protocol with every reagent or kit. The protocols each have their own Part Number and can be ordered separately, free of charge. Protocols can also be found on our website by doing a search on our Product and Service Literature System, Documents On Demand page. Alternatively, the Core does carry some commonly used protocols. Please contact the Core for more information.
Q3. Is there a User Manual for Primer Express® that I can download from the Applied Biosystems web page?
No, however there is a Tutorial Book and a Primer Express® Book that gets loaded into the Primer Express® Documents folder when the Primer Express® application is installed onto your computer. In addition, there are two tutorials on our website that can be viewed on the technical support page under Technical Tools.
Q4. I'm not sure what dye was used to label the probe in the Pre-Developed TaqMan® Assay reagents (PDAR) that I'm using. How can I find out?
PDARs are labeled with a variety of dyes, depending on their application. For a complete listing and further information on PDARs please go here. At the bottom of this page look for "4. Part Numbers Legend". This will help you determine which dye labels you have.
Q5. Why am I able to read wells containing the VIC™ dye when I am looking at the FAM dye layer?
Both VIC™ and FAM emit fluorescence in a similar range so that they will be visible in each others layers. If you are doing single dyes in a well you should not label the wells with an inappropriate dye. If you are multiplexing the two dyes together make sure that you have the spectral compensation for real-time turned on. This will tighten the range and filter out the wavelengths that are overlapping. Multiplex assay design procedures are covered in User Bulletin #5 which can be downloaded from our website.
Q6. Is it possible to run a multiplex RT-PCR on the ABI PRISM® 7000 or ABI Prism® 7900HT Sequence Detection System, and can I use more than two dyes in a single tube?
The ABI PRISM® 7000 or ABI Prism® 7900HT is capable of performing a multiplex RT-PCR assay. Currently, AB only supports assays using no more than two reporter dyes in a single assay. The analysis software has limited abilities to analyze two standard curves at one time, although two standard curves can be run on the same plate. To analyze two standard curves it is necessary to Export the data and re-plot the data in a spreadsheet program. To understand how to design a multiplex assay you should refer to User Bulletin #5: Multiplex PCR with TaqMan¨ Vic Probes. To learn how to analyze exported data in a spreadsheet program please refer to the tutorial, Generating ABI Prism® 7700 Standard Curve
Q7. What is the advantage of using VIC™ dye instead of JOE™ or HEX dye for a TaqMan® probe?
There are four main benefits:
- VIC™ dye is two to three times brighter than JOE™ or HEX. Therefore, VIC™ dye provides for better sensitivity in a TaqMan® reaction.
- VIC™ dye can be synthesized in all three synthesis scales.
- The spectral emission of probes labeled with VIC™ dye is narrower than that of JOE™ dye, therefore, there is greater distinction between labeled probes in applications that require multiplexing.
- The cost of synthesizing a VIC™ labeled probe is considerably less expensive than the synthesis with HEX and JOE.
For more information on VIC™, please refer to User Bulletin 5, Multiplex PCR with TaqMan® VIC™ Probes.
Q8. Can AmpliTaq Gold™ be used in a one-step or a two-step RT-PCR?
Yes. ABI now sell the GeneAmp® Gold RNA PCR Reagent kit (P/N 4308206), which comes with a pAW109 kit control, as well as the GeneAmp® Gold RNA PCR Core Kit (P/N 4312765) that does not contain the control reagents. If you would like more information as to how it can be used in either a one-step or two-step RT-PCR reaction, you can get a copy of the kit protocol. For ordering information, please refer to the Applied Biosystems electronic store.
Q9. Does AmpliTaq Gold™ leave an A overhang as does AmpliTaq® DNA Polymerase?
AmpliTaq Gold™ is the same enyzme as AmpliTaq® DNAPolymerase except that it is supplied in the inactive state. Once activated it has the same properties and characteristics as AmpliTaq. Therefore, since it does not have "proofreading" ability, it too will leave an A overhang. This happens arbitrarily, and as with AmpliTaq DNA Polymerase, in order to drive the reaction to the extra A state, the final extension time at 72°C should be increased to 15-30 minutes.
Q10. Does the activation of AmpliTaq Gold™ at 95°C for 10 minutes interfere with the half-life of the enzyme?
The half-life of AmpliTaq Gold™ at 95°C is 40 minutes. This is with constant incubation at the described temperature. It is a known fact that during PCR, the sample, and therefore the enzyme, is truly only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95°C is approximately 100 cycles. Example: AmpliTaq® DNA Polymerase experiences about 20 seconds (È0.33 min) at 95°C per PCR cycle. t1/2 is at least 33 minutes; (35-40 mins) therefore, 33 min / 0.33 min/cycle = 100 cycles 100 PCR cycles reduces enzyme activity by 50%.
Q11. Once AmpliTaq Gold™ is activated does it remain in an active state?
Yes, once activated, the enzyme remains active. Lowering the temperature will not inactivate AmpliTaq Gold™.
Q12. Why does amplification efficiency decrease or plateau in the late cycles?
Three limitations may contribute to the decrease in amplification efficiency:
- Plateau occurs in PCR because of the progressive reduction in the efficiency of the primer-template complex formation due to product reannealing. The primers in PCR are always present at high excess and the kinetics of reannealing are determined by the molecule in the greatest concentration. Typically, total initial PCR primer concentrations are 2x10-6 M. However, as the reaction proceeds, the DNA target concentration increases and the primer concentration decreases, albeit only slightly, to 1.4x10-7 M to 2x10-6 M, and it takes a longer time for the primer-template complex to form. If the product strands have a chance to reanneal to themselves, forming a very stable complex, the primers will not have a binding site for extension and doubling may not occur. This snap-back of product strands reannealing to themselves can occur in a few seconds (or less) at high DNA concentrations.
- Primer concentrationdepletion. One cause of primer depletion is the formation of primer artifacts and other spurious, non-specific amplification products.
- Insufficient enzyme concentration or polymerization time in late cycles. In a typical PCR amplification, 2.0 - 2.5 U or roughly 8x1010 molecules of AmpliTaq DNA Polymerase are used. In plateau there are roughly 1x1012 template copies leaving less than one molecule of AmpliTaq¨ DNA Polymerase per primer-template complex. One way to overcome the limiting polymerase is to increase the extension time in the later cycles of the PCR amplification.
Q13. What is "Hot-start" PCR?
"Hot-start" is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. The major obstacle to obtaining highly sensitive and specific amplifications appears to be competing side reactions such as the amplification of non-target sequences in background DNA (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Pre-PCR mispriming can occur at anytime all components necessary for amplification are present at permissive (or below optimal annealing) temperatures such as during reaction set up. A "hot start" can be performed either manually or can be automated utilizing AmpliTaq Gold™. In the manual "hot start" technique, a key component necessary for amplification is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. The component withheld from the reaction mix can be primers, AmpliTaq® DNA Polymerase, Magnesium Chloride, or dNTPs. The reaction mix, minus this component, is heated to a temperature slightly above the annealing temperature (60°C - 72°C). Once the reaction mix reaches this temperature, the missing component is added and the PCR amplification is allowed to proceed. Because a key component was withheld from the reaction at permissive temperatures, competing side reactions are minimized and a specific amplification occurred.
AmpliTaq Gold™ facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold™ is an inactive form of AmpliTaq® DNA Polymerase, that once activated performs just as AmpliTaq® does. Since it is provided in its inactive form, it can be added to a PCR amplification without the fear of pre-PCR misprimed primers being extended. Once all of the components for amplification have been added to a tube, the reaction is heated to 95°C for 10 minutes.
Q14. Does Applied Biosystems have a document that contains chemistry optimization guidelines for the ABI PRISM® 7000, ABI PRISM® 7900HT?
Yes, Applied Biosystems has developed TaqMan® Quick Assay Development Guidelines for the following applications: DNA and RNA quantitation, and Allelic Discrimination. The Quick Assay Development Guidelines are published in the TaqMan® Universal PCR Master Mix protocol booklet (P/N 4304449). The Universal PCR Master Mix protocol can be found here.
You can also find this information by downloading the Sequence Detection Systems Quantitative Assay Design and Optimization guide from our documentation library.
Q15. Can Relative Quantitation of Gene Expression be performed on the ABI PRISM® 7700 Sequence Detection System?
Yes, relative quantitation of gene expression can be run on the 7700. For detailed instruction please refer to User Bulletin #2: Relative Quantitation of Gene Expression. User Bulletin #2 can be ordered on our Fax-on-Demand service by calling 1-800-487-6809. Press 2 and punch in document ID# 777802.
You may also want to consult User Bulletin #5: Multiplex PCR with TaqMan ® VIC probes and the Sequence Detection Systems Quantitative Assay Design and Optimization guide from our documentation library.
Q16. What can be used to dilute my TaqMan® probe?
The probe can be diluted in 10 mM Tris-HCl, pH 8.0 (at 25°C), 1 mM EDTA. We recommend storing your probes (stocks and working solutions) at -20 °C in aliquots. This is to cut down on the number of times the probe is subject to freeze-thaws.
For more information on diluting primers and probes, please refer to our tutorial.
Q17. Does AmpliTaq Gold® DNA Polymerase contain 3'- 5' exonuclease (proofreading) activity?
No, AmpliTaq Gold does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by:
- Decreasing the final concentration of each nucleotide to 40-50 uM.
- Using the lowest MgCl2 concentration possible.
- Using less enzyme.
- Decreasing extension times.
- Using the highest annealing temperature possible.
- Using as few cycles as possible.
Q18. How does AmpliTaq Gold® DNA Polymerase differ from AmpliTaq® DNA Polymerase?
AmpliTaq Gold™ DNA Polymerase is a chemically modified form of AmpliTaq DNA Polymerase. This inactivation occurs as a result of the use of a proprietary chemical group, which binds to the active site of the enzyme. Removal of this chemical group, and thus activation of the enzyme, occurs during incubation at 95¡C. We recommend an initial activation step of 95¡C for 10 minutes when using GeneAmp™ 10X PCR Buffer and GeneAmp™ 10X PCR Buffer II and on an Applied Biosystems Thermal Cycler. If you are using the GeneAmp™ 10X PCR Gold Buffer, the initial activation time should be 95°C for 5 minutes. Each subsequent denaturing step, at 95°C, activates more of the enzyme, thus enzyme activation occurs throughout the thermal cycling reaction.
Q19. How do I use Primer Express for designing primer and probe sets for TaqMan®?
Primer and probe sets for TaqMan® can be designed by using an easy to follow tutorial for Primer Express.
Q20. Where can I find information about TaqMan® Assays and the ABI Prism® Sequence Detection Systems?
Q21. Can I use Oligo (dT)16 for my RT reactions in conjunction with the TaqMan® Human Endogenous control Plate (P/N 4309199)?
No. The reverse transcription of total RNA to cDNA must be done with random hexamers. This is because the 18S rRNA assay cannot evaluate poly A+ purified RNA samples because most of the ribosomal RNA has been removed. For more information, please refer to the protocol.
Q22. How can I download a SDS protocol?
Visit the Technical Support web page at http://www.appliedbiosystems.com/support. Selecting 'Product and Service Literature' will bring you to our document search window. In the Product Market Line window, select Sequence Detection. This will refresh the Product window and display only SDS-related products. You can now either select Protocol under Document Type, or for a more refined search, first select a category in the Product window (Taqman, SYBR, etc.). A keyword or phrase may also be entered in the Document Title / Keyword section.
Q23. What is the minimum/maximum reaction volume I should be using in the ABI PRISM® 7000 and ABI Prism® 7900HT?
For 96 wells plate, the minimum reaction volume is 25µl for real time PCR and for Allelic Discrimination plate reads. For 384 wells plate, the minimum reaction volume is 10µl for real time PCR and 5µl for Allelic Discrimination plate reads. The maximum volume for 96 wells reaction is 50µl and for 384 wells reaction is 20µl.
Q24. Can I collect my data on the PC connected to the ABI Prism® 7000/7900HT and analyze it on another PC?
Yes, there is a free standing PC in the core that is available for data analysis and primer express usage. ABI Prism® 7000 software is available for free download. SDS 2.1 (running the ABI Prism® 7900HT) is available for a charge from ABI. For more information please contact the Core.
Q25 What does the Core provide?
The Core provides equipments (currently ABI Prism® 7000 and ABI Prism® 7900HT), softwares (Primer Express, SDS 1.1 and SDS 2.1), training, experiment design consultation and troubleshooting.