SuperScript™ Choice System for cDNA Synthesis - FAQs

查看更多产品信息 SuperScript™ Choice System for cDNA Synthesis - FAQs (18090019)

5 个常见问题解答

我正在使用SuperScript Choice系统，在合成双链cDNA时出现了问题。对此您有什么好的建议吗？

我们分析的原因和建议如下：

•第一链合成产物浓度过低：起始RNA的质量过低，反应体积过大，且/或引物浓度过低或引物未针对RNA正确退火。
•第二链合成产物浓度过低：未加入RNAse H，或者在进行第二链反应时进行了不当稀释。
•克隆数量过少：最可能是接头连接/磷酸化时出现问题；请使用phiX Hae III片段作为对照；为其连接接头并进行磷酸化处理，以判定这一环节是否存在问题。

我想从mRNA生成双链DNA(dsDNA)，贵公司有适合的试剂盒吗？

当然，为此我们提供了三种试剂盒：

1.SuperScript Plasmid System，适用于cDNA合成和克隆 ◦可生成cDNA并将其克隆到含有CMV启动子、兼容Gateway的载体(pCMVSport6)之中，以便进行哺乳动物细胞表达筛选
◦实验需要mRNA（必须具有poly(A)尾）作为起始材料
◦可以在大肠杆菌中进行探针筛查，但不可进行表达筛查
◦产生单向、包含Sal I / Not I酶切位点的dsDNA

2.SuperScript Choice系统，适用于cDNA合成 ◦适用于任何RNA来源
◦可以使用随机引物、oligo(dT)引物或混合引物
◦会产生带有EcoRI末端的cDNA，可以克隆到任何具有EcoRI 位点的载体（质粒或噬菌体）中
◦有益于生成cDNA文库
◦产生的dsDNA可双向克隆

3.SuperScript双链cDNA合成试剂盒 ◦可用于生成平端、双链cDNA
◦可使用总RNA或mRNA作为起始材料

How long can I store the cDNA from my reverse transcription step?

You can store your cDNA at 2-6 degrees C for up to 24 hours. For long-term storage, store the cDNA at -15 to -25 degrees C and add EDTA to a final concentration of 1 mM to prevent degradation.

I'm using your SuperScript Choice System and am having problems with generating my double-stranded cDNA. Can you please offer your suggestions?

Please see the following suggestions:

-Low concentration of first-strand synthesis: starting RNA quality is poor or too low, reaction volume is too large, and/or primer concentration is too low or primers were not annealed to RNA properly.
-Low concentration of second-strand synthesis: RNAse H was not added or improper dilution to the second-strand volume was used.
-Low number of clones: there is most likely a problem with the adapter ligation/phosphorylation; use phiX Hae III fragments as a control; ligate adapters to it and phosphorylate to determine whether this is the problem.

I'd like to generate double-stranded DNA (dsDNA) from mRNA. Do you have a kit for this?

1.SuperScript Plasmid System for cDNA Synthesis and Cloning
-Generating and cloning cDNA into a CMV-containing Gateway-compatible vector (pCMVSport6) for mammalian screening
-mRNA required as starting material (must have poly(A) tail)
-Can probe screen in E.coli but not expression screen
-Resulting dsDNA is unidirectional with Sal I / Not I sites

2.SuperScript Choice System for cDNA Synthesis
-Will work on any RNA source
-Can use random, oligo(dT), or combination
-Will result in cDNA with EcoRI ends which can be cloned into any vector (plasmid or phage) that has EcoRI sites
-Good for generation of cDNA libraries
-Resulting dsDNA will clone bidirectionally

3.SuperScript Double-Stranded cDNA Synthesis Kit
-For generating blunt-ended, double-stranded cDNA
-Will work for total RNA or mRNA as starting material