One Shot™ Mach1™ T1 Phage-Resistant Chemically Competent E. coli - FAQs

我正在构建酵母基因组文库，并希望利用你们的感受态进行转化和扩增。选择菌株时我应该关注哪些基因型特征？

我们推荐使用mcr/mrr–菌株，这种菌株可以防止甲基化的真核DNA在大肠杆菌宿主中的扩增受到限制。我们还推荐使用T1R菌株，因为T1是基因组/cDNA文库中一种常见的污染。

生长最快的感受态细胞是哪个？

我们的Mach1-T1R感受态细胞比我们其它常规克隆菌株长得快。它的倍增时间是54分钟，而普通的克隆菌株如DH5α的倍增时间则需要增加70分钟。37摄氏度下将转化混合液铺板后8小时左右就可以在培养板上看到Mach1-T1R菌株的克隆。将过夜培养的单个大克隆接种到1.5毫升离心管37摄氏度下培养4小时就能进行质粒小抽。

I'm making a yeast genomic library and want to transform and amplify it in one of your competent cell strains. What genotype features should I look for in choosing a good strain?

We would recommend a mcr/mrr- strain, which prevents restriction of methylated eukaryotic DNA in the E. coli host. We would also recommend using a T1R strain, as T1 is a common contaminant in genomic/cDNA libraries.

Which is your fastest growing strain of competent cells?

Our Mach1-T1R competent cells grow faster than any of our common cloning strains. It has a doubling time of 54 minutes versus doubling times in excess of 70 mins for standard cloning strains, such as DH5α cells. Colonies of Mach1-T1R begin to be visible on a plate 8 hours after plating the transformation mix at 37 degrees C. It can be mini-prepped from 1.5 mL cultures in as little as 4 hours at 37 degrees C after inoculation with a single large overnight colony.

What advantages do your Stbl2 cells offer over other cloning strains?

There are other strains available that may function similarly to Stbl2 cells in stabilizing inserts or vectors with repeated DNA sequences. However, one advantage of Stbl2 cells over many similar strains is that they are sensitive to Kanamycin, so you can use Stbl2 to propagate plasmids containing a Kanamycin resistance marker. 

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.

Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.

Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

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.