SuperScript™ Plasmid System with Gateway™Technology and ElectroMAX™ DH10B Competent Cells - FAQs

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

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

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis.

The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared.

Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension.

Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.