M-MLV Reverse Transcriptase (200 U/μL)

旧バージョンのソフトウェアでは、Relative Quantification Plate(⊿⊿Ct）アッセイを行う場合、同一ファイル中に融解曲線を設定することができません。ただし最近の機種や、7500/7500fastの最新のsofware( ver.2.0.5)では、サイクルステージの直後にmelt curve(融解曲線）が設定可能となっています。 なお旧バージョンのソフトウェアの場合は、Relative Quantification Plate(⊿⊿Ct）アッセイを行った後に、新しいRunファイルを作成し、融解曲線のみのプログラムでRunを行ってください。 融解曲線のみのRunを行う場合、"File"から "New"を選択します。展開された" New Document Wizard"画面中の"Assay"のプルダウンメニューから"DISSOCIATION"を選択し、融解曲線のみのRunを行ってください。この場合、解析ファイルとは別に融解曲線のみのSDSファイルが作成されます。

While the volume is dependent on the starting amount of RNA used for the first-strand synthesis and the abundance of the target gene, we'd recommend starting with 10% of the first-strand reaction for your PCR reaction.

The optimal temperature for SuperScript III RT is 50 degrees C, and can be used up to 55 degrees C. For some qRT-PCR reactions where gene-specific primers are used, you can do the RT reaction at 60 degrees C. The optimal temperature for SuperScript II RT is 42 degrees C, and can be used up to 50 degrees C. Optimal temperature for MMLV is 42 degrees C. ThermoScript RT shows optimal activity at 60 degrees C, and can be used at temperatures as high as 70 degrees C (for amplicons expected to be 1 kb or less). For PCR products expected to be greater than 1 kb, a maximum first strand synthesis temperature of 60-65 degrees C is suggested. Be sure your first-strand primer anneals at the high temperature, especially when gene-specific primers are used for high-temperature stable reverse transcriptases. We recommend oligo (dT)20 for cDNA synthesis when using an oligo (dT) primer for first-strand synthesis with these enzymes.

Yes, you can use a DNA-RNA hybrid as a template for M-MLV Reverse Transcriptase.

We have not tested this for SuperScript reverse transcriptases, so we cannot guarantee it would also work with those products.

This article can be used as a reference for additional information.

Find additional tips, troubleshooting help, and resources within our Reverse Transcription and RACE Support Center.

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).