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A registration key is a one-of-a-kind ID generated by the FME Licensing Assistant from system data. It's Safe's way of limiting a single fixed license to a single computer.
2: What is a registration key number?
A registration key is a code of letters and numbers that allows access to one of the many Thomson Reuters products, such as Westlaw, CLEAR, Firm Central, and more.
3: What is the registration key?
Each person will create an individual user account by entering the customer's account number, an online registration key (available from your local dealer), and basic billing and shipping address information. The account administrator will be the first account created.
Curves are a very versatile and precise color correction tool. The native After Effects version lacks control and accuracy to take full advantage of it. Fresh Curves makes up for these drawbacks so you can get the most out of this basic tool. The integrated histogram helps you find and modify desired areas and gives feedback to your changes. Fresh Curves has brightness, saturation and hue channels for more direct control on top of the usual RGBA. It comes in handy for those out of range float colors or when fiddeling with details. It includes invert curves which you can work in your own color space. Apply your custom “gamma curve”, do the work and apply and the inverted one.
Curves are a very versatile and precise color correction tool. The native After Effects version lacks control and accuracy to take full advantage of it. Fresh Curves makes up for these drawbacks so you can get the most out of this basic tool.
Obtaining high quality and intact RNA is the first and often the most critical step in performing RT-PCR and real-time PCR. RNA is easily degraded since RNase is very hard to inactivate. Several precautions need to be taken to prevent RNA from degradation. People should always wear a clean lab coat, disposable gloves, and change gloves frequently. The bench should be clean. Any aqueous solutions, tubes, and pipettes used for the procedure should be sterile and RNAse-free. To avoid contaminating your sample with RNases, do not talk while processing RNA extraction.
Reverse transcription involves the synthesis of DNA from RNA by using an RNA-dependent DNA polymerase, the reverse of normal transcription, which is from RNA to DNA. Although there are many kits commercially available for RT, the reverse transcriptase used in those kits usually is M-MLV reverse transcriptase from the Moloney murine leukemia virus or AMV reverse transcriptase from the avian myeloblastosis virus. M-MLV reverse transcriptase is the preferred reverse transcriptase in cDNA synthesis for long messenger RNA (mRNA) templates (>5 kb) because the RNase H activity of M-MLV reverse transcriptase is weaker than the commonly used AMV reverse transcriptase (2). Since M-MLV reverse transcriptase is less processive than AMV reverse transcriptase, therefore, more units of the M-MLV enzyme are required to generate the same amount of cDNA as in the AMV reaction (2). The following are the basic procedures for RT using M-MLV reverse transcriptase according to the manufacturer’s instruction.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase. There are many DNA polymerases commercially available. Although their efficiency may be different, they are suitable for regular RT-PCR to determine the expression level of mRNA. Some experiments which will use PCR products for cloning purposes, especially those for cloning of promoter region with high G-C content, need to use high fidelity DNA polymerase. The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The following is an example of a PCR performed in our laboratory.
Traditional PCR uses agarose gel for detection of PCR amplification at the final phase or endpoint of the PCR (plateau). However, real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction. Theoretically, there is a quantitative relationship between the amount of starting target sample and the amount of PCR product at any given cycle number. Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured at the exponential phase of the PCR, which makes quantitation of DNA and RNA easier and more precise than traditional methods. There are two methods, which are often used in the laboratory. One is the 5′ nuclease assay in which an oligonucleotide called a TaqMan® Probe is added to the PCR reagent master mix. This probe is designed to anneal to a specific sequence of template between the forward and reverse primers and is also designed with a high-energy dye termed a Reporter at the 5′ end, and a low-energy molecule termed a Quencher at the 3′ end. When this probe is intact and excited by a light source, the Reporter dye’s emission is suppressed by the Quencher dye as a result of the close proximity of the dyes. When the probe is cleaved by the 5′ nuclease activity of the enzyme, the distance between the Reporter and the Quencher increases causing the transfer of energy to stop. The fluorescent emissions of the reporter increase and the quencher decrease. An increase in Reporter fluorescent signal is directly proportional to the number of amplicons generated. Another real-time PCR method is by using SYBR Green Dye, which can bind the minor groove of any double-stranded DNA molecule. When SYBR Green dye binds to double-stranded DNA, the intensity of the fluorescent emissions increases. As more double-stranded amplicons are produced, SYBR Green dye signal will increase. The following is an example of real-time PCR by using iQ™ SYBR Green Supermix and performed on an iQ5 multicolor real-time PCR detection system.
Currently, there are a number of RNA isolation kits commercially available. Although it is convenient, time-saving, and avoids contact with phenol/chloroform using commercially available kits, those kits using silica-membrane spin columns may not be ideal for studies of insoluble nanoparticle since the nanoparticles may clog the membrane pore of the spin column. Therefore, it is important to choose the right reagents or kits for total RNA isolation according to different experiments and specific characteristics of different nanoparticles. In our laboratory, we use TRI Reagent to isolate total RNA for nanoparticle studies. TRI Reagent is a mixture of guanidine thiocyanate and phenol in a monophase solution, which can effectively dissolve DNA, RNA, and protein after homogenization or lysis of tissue samples. It performs well with large or small amounts of tissue or cells. Here, we describe the procedures for isolating total RNA using TRI Reagent according to the manufacturer’s instructions.
- Fresh Curves has brightness, saturation, and hue channels for more direct control on top of the usual RGBA.
- Now it’s possible to adjust on attributes based on another. Relative Curves also have a built-in pre blur and a post blur curve.
- With tangent handles for more curve control. Interpolation modes are similar to the ones in the AE graph editor round things off.
- Using invert you can work in your own color space. Apply your custom “gamma curve”, do the work, and apply and the inverted one.
The integrated histograms help you find and modify desired areas and gives feedback to your changes.
- Tired of that old, tiny interface? Here is a big, nice resizeable one.
- Will come in handy for those out of range float colors or when fiddling with details.
- Need to be precise? Just edit or sample your values. And change the color range to what you are comfortable with.
1: Click Install Key after navigating to Tools & Settings > License Management > Plesk License Key.
2: Choose Upload a licence key file.
3: Click OK after providing the path to the key file you downloaded from the email.