Is there a User Manual for Primer Express® software?
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
Are there any TaqMan® Endogenous Controls reagents available that can be used with rat samples?
We have TaqMan® Endogenous Controls for 18S Ribosomal RNA and a kit (Cat. No. 4308313) that can be used with rat samples. The Eukaryotic 18S Ribosomal RNA Reagent is in a highly conserved area that allows this TaqMan® Endogenous Control to be used for rat, human and mouse. In addition, we have newer TaqMan® Gene Expression Rat Endogenous Controls, including beta glucuronidase and beta-2 microglobulin. These can be found by searching our Gene Expression Assays and specifying the species of interest as R. norvegicus and the set membership as Endogenous Controls.
Answer Id: 1006
What is the difference between MultiScribe™ Reverse Transcriptase and MuLV Reverse Transcriptase?
MultiScribe™ Reverse Transcriptase (Cat. No. 4311235) and MuLV Reverse Transcriptase (Cat. No. N808-0018) are the same enzyme, a recombinant Moloney Murine Leukemia Virus (MuLV) Reverse Transcriptase. The only difference is in the recommended usage for each enzyme. MultiScribe™ Reverse Transcriptase is recommended for use with quantitative nuclease assays, and MuLV is recommended for use in traditional RT-PCR assays.
Answer Id: 1008
What is the advantage of using VIC® dye as a reporter instead of JOE™ dye for a TaqMan® probe?
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
Does AmpliTaq® DNA Polymerase have reverse transcriptase activity?
Does AmpliTaq® DNA Polymerase have exonuclease activities?
AmpliTaq® DNA Polymerase lacks a 3' - 5' exonuclease activity. However, the enzyme does have a fork-like, structure-dependent polymerization-enhanced 5 ' - 3' nuclease activity. During the extension step of a PCR amplification, the enzyme will hydrolyze any blocking strand starting from its 5' end, replacing the lost material by extending the new chain.
Answer Id: 1073
Will PCR amplifications previously successful with regular AmpliTaq® Polymerase work as successfully with AmpliTaq Gold® Polymerase?
They will work more successfully and reproducibly with AmpliTaq Gold® Polymerase. Although AmpliTaq Gold® Polymerase is the same exact enzyme as AmpliTaq® Polymerase, the fact that the reaction is being driven towards high specificity and yield may require some modifications to previous conditions. For example, if the previous reaction was on the edge of optimization, magnesium chloride concentrations may need to be re-optimized or if previous reactions were being run in a pH suboptimal for AmpliTaq Gold® Polymerase, reaction conditions and sample preparation protocols may need to be revisited. Activation time for AmpliTaq Gold® Polymerase will also need to be determined empirically and is dependent on cycler type.
Answer Id: 1074
Can AmpliTaq Gold® Polymerase be used in a one-step or a two-step RT-PCR?
Yes. We have the GeneAmp® Gold RNA PCR Reagent Kit (Cat. No. 4308206), which comes with a pAW109 kit control, as well as the GeneAmp® Gold RNA PCR Core Kit (Cat. No. 4312765) that does not contain the control reagents. For more information on how each kit can be used in either a one-step or two-step RT-PCR reaction, please refer to the GeneAmp® Gold RNA PCR Reagent Kit Protocol.
Answer Id: 1075
Why is AmpliTaq Gold® Polymerase the enzyme of choice for multiplex PCR?
Multiplex PCR involves the coamplification of multiple amplicons in a single PCR. Since multiple sets of primers are being added to a single reaction, the potential for primer dimer formation as well as a general loss of specificity and a decreased yield of specific product exists. The ability to control the activation of AmpliTaq Gold® Polymerase via hot start, so that the multiple primers do not have the possibility to react with themselves, has proven successful at alleviating these complications and dramatically increasing specific product yield.
Answer Id: 1076
Do AmpliTaq® DNA Polymerase and AmpliTaq® Gold® DNA Polymerase add on extra A to the PCR product?
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 the activation of AmpliTaq Gold® DNA Polymerase at 95 degrees C for 10 min interfere with the half-life of the enzyme?
The half-life of AmpliTaq Gold® Polymerase at 95 degrees C is 40 minutes. This is with constant incubation at the described temperature. During PCR, the reaction is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold® Polymerase at 95 degrees C is approximately 100 cycles.
Example: AmpliTaq Gold® DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t1/2 is at least 35 minutes; (35-40 min). Therefore, 35 min/20 sec/cycle = 105 cycles. 105 PCR cycles reduces enzyme activity by 50%.
Answer Id: 1078
Does AmpliTaq Gold® DNA Polymerase remain in an active state once it is activated?
What thermal stable DNA polymerase is recommended for PCR amplification of long PCR targets?
Successful amplification of long PCR targets is dependent variables such as sufficient extension time during the PCR amplification, cosolvent addition, pH of the reaction buffer, salt concentration, primer design, use of a hot start, DNA sample integrity, and the enzyme's proofreading and polymerase activities. Routine amplification of targets up to 5 kb has been successful with the use of AmpliTaq® Polymerase and AmpliTaq Gold® DNA Polymerase under standard PCR buffer conditions. However, for targets ranging from 5 kb up to 40 kb, rTth DNA Polymerase, XL works optimally. This enzyme and its optimized buffer promote not only efficient DNA synthesis, but also allow for correction of nucleotide misincorporations that might otherwise prematurely terminate synthesis. Please refer to the GeneAmp® XL PCR Kit Product Insert for more information.
Answer Id: 1083
When amplifying long PCR targets, is the concentration of the deoxynucleoside triphosphates (dNTPs) limiting?
The concentration of dNTPs in a standard PCR amplification is 200 μM each, for a total of 800 μM. This total dNTP amount corresponds to 39 μg of dNTPs. This is a huge excess and, when generating long PCR fragments, is not a limiting factor during the PCR amplification, as the amount of target DNA generated is generally no more than 1 μg. More importantly, the reaction condition variables need to be monitored more closely in order for successful long PCR amplification to occur.
Answer Id: 1084
Describe the amplified product produced by the control reagents in the GeneAmp® Gold PCR Reagent Kit with Multiplex Control (Cat. No. 4312778).