DH5α Competent Cells for Subcloning - FAQs

我可将感受态细胞存储在液氮中吗？

我们不建议将感受态细胞保存在液氮中，因为极端温度会损害细胞。另外，装感受态细胞的塑料管子可能承受不了如此低的温度，从而发生破裂。

我应该如何存储我的感受态E. Coli？

我们推荐将感受态细胞存储在-80摄氏度。高于这个温度，即使存储时间很短，也会显著降低其转化效率。

我得到了过度生长的克隆，这是什么原因所致？

确保您使用了正确的抗生素和合适的浓度。另外，确保抗生素没有过期。如果克隆的形态和预期不同，那么污染可能是一个原因。检测您的SOC培液和LB培液。

我得到了白色克隆，但是克隆内都没有插入片段。我应该怎么解决这一问题？

以下是一些建议：

•小片段/接头而不是您的插入片段被克隆进了您的载体。要解决这一问题，请在连接前对插入片段进行凝胶纯化。
•确保使用了正确的X-gal和/或IPTG（当载体带有lacIq标志物时）浓度。
•在平板上涂布X-gal和/或IPTG时，留出足够的时间让试剂充分扩散进入平板。
•孵育平板足够长的时间以确保颜色完全显现。

我的平板上没有长出克隆。您可以帮助我解决这一问题吗？

我们建议尝试以下措施：

•进行pUC19转化对照；这可以告诉您所用感受态细胞的转化能力信息。
•检查平板的过期时间以及是否使用了正确的培液（LB/琼脂）。
•确认使用了正确的抗生素并且浓度也是对的。

你们是用哪种培养基来储存感受态细胞的？

我们的所有感受态细胞的制作程序和相关配方都是保密的。所有化学感受态细胞都是保存在一种含有盐混合物及一种冻存稳定剂（比如甘油或DMSO）的水溶液中的。

对于蓝白斑筛选你们有什么建议？

1.使用pUC或基于pUC的载体，这些载体含有lacZ基因的一部分，可以进行α补偿。
2.选择一种带有 lacZdeltaM15标记的E. Coli菌株。
3.将转化混合液涂布在含有X-gal的平板上。在一个100 mm平板上涂布50 µg的2% X-gal或者100微升2% bluo-gal （均可溶解于DMF或50:50的DMSO：水混合物中）并晾干。此外，也可在倒板前直接向冷却后的培液中加入终浓度50 µg/mL的X-gal或300 µg/mL的bluo-gal。平板在4摄氏度可稳定保存4周。
4.如果菌株带有lacIq标志，则需加入IPTG以诱导lac启动子。在100 mm平板上涂布30 µl的100 mM IPTG（溶于水中）。此外，也可以在倒板前直接向冷却后（大约59度）的培养液中加入IPTG至终浓度1 mM。平板在4摄氏度可稳定保存4周。
5.如果要使用 X-gal或bluo-gal进行蓝白斑筛选，那么不要将E. coli 涂布在含有葡萄糖的培养基上。因为葡萄糖会作为底物和X-gal或bluo-gal竞争从而阻止细胞变蓝。

Can I store my competent E. coli in liquid nitrogen?

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

How should I store my competent E. coli?

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

I'm getting overgrowth of colonies. Why?

Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.

I'm only getting white colonies, but none of the clones have an insert. What can I do?

Here are a few suggestions:

- Small fragments/linkers are cloning in to your vector instead of your insert; to correct this, gel-purify the insert before ligation
- Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used
- If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate
- Incubate your plate for a longer period to ensure full color development

I'm getting no colonies at all on my plates. Can you help?

We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.

What is the storage media for the competent cells?

Preparation procedures and formulations for all of our competent cells are proprietary. All chemically competent cells are delivered in an aqueous solution that contains a mixture of salts, along with a freezing stabilizer such as glycerol or DMSO.

What suggestions can you make for blue/white screening?

1. Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for ? complementation.
2. Select an E. coli strain that carries the lacZdeltaM15 marker.
3. Plate transformations on plates containing X-gal. Spread 50 µg of 2% X-gal or 100 microliters of 2% bluo-gal (both can be dissolved in DMF or 50:50 mixture of DMSO:water) on the surface of a 100 mm plate and let dry. Alternatively, add directly to the cooled medium (~50 degrees C) before pouring the plates at a final concentration of 50 µg/mL for X-gal and 300 µg/mL for bluo-gal. Plates are stable for 4 weeks at 4 degrees C.
4. If the strain used carries the lacIq marker, add IPTG to induce the lac promoter. Spread 30 µl of 100 mM IPTG (in water) on 100 mm plates. Alternatively, add the IPTG directly to cooled medium (~50 degrees C) before pouring the plates to a final concentration of 1 mM. Plates are stable for 4 weeks at 4 degrees C.
5. Do not plate E. coli on medium containing glucose if using X-gal or bluo-gal for blue-white screening. Glucose competes as a substrate and prevents cells from turning blue.

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.