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SuperScript™ Indirect cDNA Labeling Module, no purification columns - FAQs

View additional product information for SuperScript™ Indirect cDNA Labeling Module, no purification columns - FAQs (L101403)

4 product FAQs found

ABI PRISM 7000　および Applied Biosystems 7300、7500、7500、7900HT SystemのSDS ソフトウェアでは、"Relative Quantification Plate Assay（ddCt)”を指定した場合、なぜ"ADD DISSOCIATION CURVE"ボタンが無効になってしまうのですか?

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。

Can the amino-labeled DNA generated with your SuperScript Indirect Labeling kit be ethanol precipitated and stored overnight? Do you know if the amino-labeled DNA will be stable over that time?

Yes, the amino-labeled cDNA should be stable if stored precipitated in ethanol at -20 degrees C.

What is the ratio of modified/unmodified dNTPs in your SuperScript Indirect Labeling Kit?

The ratio of nucleotides is one of the key features in the optimized performance of this product, and therefore is considered proprietary. Unfortunately, we are unable to provide this information.

Can I use the SuperScript Indirect cDNA Labeling System for bacterial samples?

We have not used bacterial RNA for the validation of the SuperScript Indirect cDNA Labeling System, however the kit should work reasonably well with few modifications to the protocol.

For total RNA samples, Oligo(dT)20 primers should be used. As the manual suggests, random hexamers should be used only with mRNA or with the RNA ladder, which is included as a control. However, in the case of bacterial RNA, longer random primers should be used instead of the random hexamers included in the kit. 18mers should work well, although the temperature of reverse transcription may need to be slightly adjusted in order to obtain optimal cDNA synthesis. The reason for using longer random primers is that the rRNA in bacterial samples will also be used as template for reverse transcription, and the target cDNA will be more complex than the corresponding cDNA primed with the Oligo(dT)20. Therefore, when doing hybridizations, you can expect some degree of cross-hybridization to occur. Nevertheless, the use of long random 18mers is common practice among microarray users.