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View additional product information for One Shot™ ccdB Survival™ 2 T1R Competent Cells - FAQs (A10460)
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我们提供One Shot ccdB Survival 2 T1R 细胞（货号A10460）以用于繁殖含有ccdB基因的质粒。
所有供体载体和目标载体均含有ccdB细胞致死基因以降低非重组BP/LR质粒的背景。因此，扩增非重组质粒需要使用对ccdB致死作用具有抗性的特殊细胞（One Shot ccdB Survival 2 T1R感受态细胞）。另一方面，通用的E.coli克隆菌株，包括TOP10或DH5a，可以用于BP或LR反应转化，或用于扩增和维持重组后的Gateway重组子。
请使用我们的ccdB Survival 2 T1R感受态菌株（货号A10460）。
We offer One Shot ccdB Survival 2 T1R cells (Cat. No. A10460) for propagating ccdB-containing plasmids.
All Donor vectors and Destination vectors contain the ccdB cell death gene to reduce background of non-recombined BP/LR plasmids. Therefore, growing non-recombined vector requires special cells (One Shot ccdB Survival 2 T1R Competent Cells) which are resistant to the lethal effects of ccdB. On the other hand, general E. coli cloning strains including TOP10 or DH5a may be used for plating the BP or LR reaction, or for propagation and maintenance of recombined Gateway constructs.
Please use our ccdB Survival 2 T1R competent cell strain.
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.
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.