How does the accuracy and sensitivity of the Qubit® Quantitation Platform compare to a microplate reader?
Product FAQ
The accuracy and sensitivity of the Qubit® Quantitation Platform is the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit® kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.
Answer Id: 5020
Is there a User Manual for Primer Express® software?
Product FAQ
No. However, there is a Primer Express® User Manual and an Applications Tutorial that gets loaded into the Primer Express® software folder when the software is installed onto your computer. In addition, there is the Getting Started Guide: Primer Express® Software Version 3.0 and tutorials available on the Primer Express product page.
Answer Id: 1000
What is the advantage of using VIC® dye as a reporter instead of JOE™ dye for a TaqMan® probe?
Product FAQ
The main benefits are the following: 1) VIC® dye is two to three times brighter than JOE™ dye. Therefore, VIC® dye provides for better sensitivity in a TaqMan® reaction. 2) VIC® dye can be synthesized in all three synthesis scales. 3) 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.
Answer Id: 1052
Do AmpliTaq® DNA Polymerase and AmpliTaq® Gold® DNA Polymerase add on extra A to the PCR product?
Product FAQ
Both AmpliTaq Gold® DNA polymerase and AmpliTaq® DNA Polymerase lack proofreading activity, so they will often leave a 3'-overhang. The base most often left is a 3'-A, however, the extra base appears to be sequence dependent and one cannot always rely on the fact that even just a single base has been left. In many cases, this artifact has been useful with TA Cloning® kits. In order to drive the reaction to the extra A state, a final extension time at 72°C should be increased to 15-30 minutes.
Answer Id: 1077
Does AmpliTaq Gold® DNA Polymerase remain in an active state once it is activated?
Product FAQ
Yes, once activated, AmpliTaq Gold® DNA polymerase remains active. Lowering the temperature will not inactivate AmpliTaq Gold® DNA polymerase.
Answer Id: 1079
Why does amplification efficiency decrease or plateau in the late cycles?
Product FAQ
Three limitations may contribute to the decrease in amplification efficiency:
1. 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 2x10E-6 M. However, as the reaction proceeds, the DNA target concentration increases and the primer concentration decreases, albeit only slightly, to 1.4x10E-7 M to 2x10E-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.
2. Primer concentration depletion. One cause of primer depletion is the formation of primer artifacts and other spurious, non-specific amplification products.
3. Insufficient enzyme concentration or polymerization time in late cycles. In a typical PCR amplification, 2.0 - 2.5 U or roughly 8x10E10 molecules of AmpliTaq® DNA Polymerase are used. In plateau there are roughly 1x10E12 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.
Answer Id: 1096
What is "Hot-start" PCR?
Product FAQ
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 (mis-priming) and primer oligomerization. In an otherwise optimized PCR amplification, most non-specific products can be attributed to pre-PCR mispriming. Mispriming can occur any time all components necessary for amplification are present at permissive temperatures (below optimal annealing temperature) such as during reaction set up. A hot start can be performed either manually or can be automated utilizing AmpliTaq Gold DNA Polymerase.
In the manual hot-start technique a key component necessary for amplification, such as the enzyme, is withheld from the reaction mix until the reaction reaches a temperature above the optimal annealing temperature of the primers. Once this temperature is reached, 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 specific amplification occurs.
AmpliTaq Gold® DNA Polymerase facilitates the automation of the hot start technique and decreases the potential for contamination. AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase. Once activated, AmpliTaq Gold® DNA Polymerase performs just as AmpliTaq DNA Polymerase does. Since it is provided in its inactive form, it can be added to a reaction 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 95C for 5 - 10 minutes. This incubation activates the enzyme and allows the reaction to proceed normally.
Answer Id: 1098
Do you have a document that contains chemistry optimization guidelines for your Real-Time PCR Systems?
Product FAQ
Yes, Applied Biosystems has developed TaqMan® Rapid Assay Development Guidelines for the following research applications: DNA and RNA quantitation, and Allelic Discrimination. The Rapid Assay Development Guidelines are published in the Universal PCR Master Mix protocol (P/N 4304449).
Answer Id: 1126
In the TaqMan® Rodent GAPDH Control Reagents kit (P/N 4308313), what rodent is the Control RNA isolated from?
Product FAQ
The control RNA is total RNA isolated from a mouse lymphoma cell line.
Answer Id: 1128
Can I use elevated temperatures for reverse transcription with Moloney Murine Leukemia Virus (M-MLV)?
Product FAQ
When using GeneAmp® RNA PCR kit (part number N808-0017) with M-MLV, the recommended temperature for reverse transcription is 42°C. It is not recommended that reverse transcription be done at a higher temperature with this enzyme because it will start to lose significant activity. If it is necessary to do reverse transcription at a higher temperature due to strong secondary structure or high G+C content, the recommended enzyme to use would be rTth DNA polymerase, which is included in the GeneAmp® Thermostable rTth Reverse Transcriptase RNA PCR kit (part number N808-0069).
Answer Id: 1329
Does AmpliTaq® Gold DNA Polymerase contain exonuclease (proofreading) activity?
Product FAQ
No, AmpliTaq® Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.
Answer Id: 1338
What type of Real-Time PCR (Sequence Detection System) information is available from Applied Biosystems?
Product FAQ
Tutorials demonstrating introductions to Real-Time PCR, guidelines for designing real-time assays using Primer Express® software, and various instrument set-ups can be found under Life Technologies University on our website. User bulletins, manuals and Product inserts, protocols can be found as downloadable pdf files on our Technical Resources page.
Answer Id: 1359
Can Oligo d(T)16 RT primers be used with the TaqMan® Human Endogenous Control Plate (P/N 4309199)?
Product FAQ
No. The reverse transcription of total RNA to cDNA must be done with random hexamers or random primers (or a mixture of random and oligo dT primers) because the 18S rRNA assay cannot evaluate poly A+ purified RNA samples as most of the ribosomal RNA has been removed. The plate has been designed to evaluate only total RNA.
Answer Id: 1360
What types of templates can be used with the TaqMan® Human Endogenous control Plate (P/N 4396840)?
Product FAQ
The plate amplifies mRNA contained within total RNA. Non-human templates are not recommended, as all the assays are human specific with the exception of 18S rRNA and the internal positive control. Additionally, Poly A+ RNA cannot be accurately evaluated by the 18S rRNA assay as most of the rRNA has been removed.
Answer Id: 1361
When designing probes to perform Allelic Discrimination (SNP assays), are there any alternatives to using probes longer than 30bp in order to get the appropriate melting temperature?
Product FAQ
Applied Biosystems® TaqMan® MGB (minor groove binder) probes would be appropriate for Allelic Discrimination/SNP Genotyping probe designs. TaqMan® MGB probes have a minor groove binder at the end of the probe. This minor groove binder increases the Tm of probes, allowing the use of shorter probes. Consequently, the TaqMan ®MGB probes exhibit greater differences in Tm values between matched and mismatched probes, which provides more accurate allele discrimination.
For information on the design of TaqMan® MGB probes for Allelic Discrimination/SNP assays using Primer Express® software, please refer to our guide "Designing TaqMan® MGB Probe and Primer Sets for Allelic Discrimination Assays Using Primer Express".
Answer Id: 1365
