MultiShot™ TOP10 Chemically Competent E. coli - FAQs

使用Zeocin抗生素筛选E. Coli时有什么特别需要考虑的问题吗？

极端pH值和/或者高离子强度会抑制Zeocin抗生素的活性。为获得理想的Zeocin抗生素筛选活性，E. coli生长培养基中的盐浓度必须低于 110 mM 并且pH值必须为7.5。特别注意的是，进行Zeocin抗性筛选时应该使用低盐LB配方（每升含5 g或更少的NaCl）。

另外，任何包含完整Tn5转座元件的E. Coli菌株（比如DH5αF'IQ, SURE, SURE2）都编码ble (bleomycin)抗性基因。这些菌株具有Zeocin抗生素抗性。为了用Zeocin抗生素进行最有效的筛选，请使用不含Tn5基因的E. Coli菌株（比如TOP10, DH5a, DH10B等）。

Are there any special considerations when using Zeocin for selection in E. coli?

Extremes in pH and/or high ionic strength will inhibit the activity of Zeocin. To optimize selection in E. coli, the salt concentration in the growth medium must be< 110 mM and the pH must be 7.5. In particular, a low salt LB formulation should be used for Zeocin selection (containing 5 g or less of NaCl per Liter).

Also, anyE. coli strain that contains the complete Tn5 transposable element (i.e. DH5?F'IQ, SURE, SURE2) encodes the ble (bleomycin) resistance gene. These strains can confer resistance to Zeocin. For the most efficient selection, use anE. coli strain that does not contain the Tn5 gene (i.e. TOP10, DH5a, DH10B, etc.).

Could EDTA decrease transformation efficiency in E. coli?

Yes, the presence of EDTA could decrease transformation efficiency. It is advisable to heat inactivate ligations at 65°C for 15 minutes rather than inactivate by addition of EDTA.

Should I increase the heat shock time for my chemically competent cells during the transformation of a larger volume?

The recommended heat shock time does increase slightly with increasing volume of competent cells. For a 50 µl reaction volume, you should heat shock at 42°C for 30 seconds. For 100 µl, 45 seconds is recommended and for 250 µl, 60 seconds. It is important to do a positive control transformation of pUC19 along with transformation of your ligation product to accurately determine your relative efficiency of transformation.

Could the efficiency of my transformation in E. coli improve if I use more than the recommended amount of DNA?

It may be surprising, but in most cases transformation efficiency per µg of DNA will actually decrease when higher amounts of plasmid are transformed in one reaction. While you may see more colonies on your plates, much of the extra plasmid DNA you added will actually be wasted. Competent cells eventually become oversaturated with DNA, and adding more plasmid beyond that level will not result in any additional colonies. For example, when transforming 10 pg plasmid DNA, the efficiency of TOP10 cells is 1.0x10E9 colonies per µg of DNA that you added. If you transform 1 ng all at once, the overall efficiency is likely to decrease to ~1.0x 10E8 colonies per µg, and transforming 1 µg in a single reaction will likely result in efficiency less than 1.0 x 10E6 colonies per µg.

To maximize colony yield, it is better to transform smaller amounts of DNA in multiple reactions rather than adding all of the DNA to one reaction. This is most important when transforming a library, where you ideally want each plasmid to be represented by a colony after transformation.