Subcloning Efficiency™ DH5α 感受态细胞

如果插入片段对于宿主细胞有潜在毒性，您可以尝试以下建议：

•转化TOP10或DH5α细胞后，在25-30摄氏度而不是在37摄氏度下孵育。这会降低生长速度并能提高克隆具有潜在毒性的插入片段的几率。
•尝试使用TOP10F’细胞进行转化，但是不在培养板中加入IPTG。这些细胞带lacIq阻抑物抑制从lac启动子起始表达，因此能克隆毒性基因。请注意，没有加入IPTG时不能进行蓝白斑筛选。
•尝试使用Stbl2细胞进行转化。

有少数例外，但他们主要的区别在于所保证的转化效率：

Subcloning Efficiency细胞每转化1 µg pUC19或pUC18超螺旋质粒保证产生至少1.0 x 10E6个转化子。
Library Efficiency细胞每转化1 µg pUC19或pUC18 DNA保证产生至少1.0 x 10E8个转化子。
MAX Efficiency细胞每转化1 µg pUC19或pUC18 DNA保证产生至少1.0 x 10E9个转化子。

If the insert is potentially toxic to the host cells, here are some suggestions that you can try:

- After transforming TOP10 or DH5? cells, incubate at 25-30°C instead of 37°C. This will slow down the growth and will increase the chances of cloning a potentially toxic insert.
- Try using TOP10F' cells for the transformation, but do not add IPTG to the plates. These cells carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. Keep in mind that in the absence of IPTG, blue-white screening cannot be performed.
- Try using Stbl2 cells for the transformation.

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.

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:

Subcloning Efficiency cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA