One Shot™ TOP10 Chemically Competent E. coli, 11 x 50 μL - FAQs

TOP10细胞与TOP10F’细胞的区别是什么？

TOP10与TOP10F’细胞的区别仅在于后者包含F’游离体因而带有四环素抗性基因，而且能够从带有f1复制起点的载体菌株中分离单链DNA。F’ 游离体同时还带有lacIq抑制子，因此可用IPTG诱导trc、ta、和lac启动子的表达。TOP10F’细胞需要IPTG诱导进行蓝白斑筛选。

我正在尝试克隆一个毒性可能非常大的插入片段。我使用过DH5α和TOP10细胞进行转化但是没有在平板上得到克隆。你们能为我提供一些建议吗？

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。

你们的大肠杆菌菌株是源于K12菌株吗？

我们大部分大肠杆菌菌株是源于K12菌株。例外的情况包括BL21菌株（源于大肠杆菌B，这一菌株也被认为是非致病性的）、Mach1（源于大肠杆菌W）及HB101。HB101源于K12/大肠杆菌B的杂交株。详见 FOCUS, 11:3, p. 56.

What is the difference between TOP10 and TOP10F' cells?

The only difference between TOP10 and TOP10F' cells is that the latter contain the F' episome that carries the tetracycline resistance gene and allows isolation of single-stranded DNA from vectors that have an f1 origin of replication. The F' episome also carries the lacIq repressor for inducible expression from trc, tac, and lac promoters using IPTG. TOP10F' cells require IPTG induction for blue/white screening.

I am trying to clone an insert that is supposedly pretty toxic. I used DH5? and TOP10 cells for the transformation and got no colonies on the plate. Do you have any suggestions for me?

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.

What generation is your ViraPower lentiviral expression system? Can I use it with a 2nd generation lentiviral packaging mix?

Our ViraPower lentiviral expression system is a 3rd generation system with regard to safety features. Our lentiviral expression vectors are derived from wild type HIV, but nearly all the wild type viral proteins (e.g., Vpr, Vpu, Vif, Nef, Tat) have been removed and the HIV envelope is not used. VSV-G (vesicular stomatitis virus G) envelope protein is used instead. Our ViraPower lentiviral expression system can be used with a 2nd generation lentiviral packaging mix. However, our lentiviral packaging mix would not be compatible with a 2nd generation lentiviral expression vector.

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.

I need a competent cell line that will generate as much DNA as possible from my vector of interest. What do you recommend?

The three most important things in this case are: endA- genotype, a high copy number origin of replication, and the culture scale. In addition, make sure (a) the culture is grown at 37 degrees C (if lower, the copy number can be lower), (b) try a super-rich media, like Terrific broth, (c) aerate the culture well (the volume of media should be no more than 10% of the volume of the flask), and (d) shake at 200-250 rpm.

Can I directly clone, propagate and express in BL21 without using TOP10?

It is imperative that a cloning strain such as TOP10 be used for characterization of the plasmid, propagation, and maintenance. BL21 cells are wild-type for endA and recA, which could result in poor miniprep quality and a greater chance of plasmid rearrangements due to recombination. In addition, BL21 cells contain the T7 RNA polymerase gene which is expressed at low levels even in the absence of inducer. If the gene is toxic to E. coli, plasmid instability and/or cell death can result.

I need to clone unmethylated DNA from a PCR reaction using a strain that has the hsdRMS mutation to avoid restriction after transformation. Is TOP10 suitable for my purposes?

Yes, TOP10 has the hsdRMS mutation, so this strain can be used to clone DNA from PCR reactions and other non-methylated sources. hsdRMS is a mutation in the system that E. coli uses to recognize foreign DNA. There are two parts to this system, methylation and restriction. E. coli methylate DNA at certain sequences, and if the DNA is not methylated at these sequences it will be recognized as foreign and restricted. Thus, if unmethylated DNA is transformed into E.coli that does not carry the hsdRMS genotype, it is recognized as foreign and enzymatically degraded.

What is the reason for the nupG mutation in the genotypes for TOP10- and DH10B-related E. coli strains?

nupG is a mutation for the transport of nucleosides. The nupG site is next to endA on the chromosome, and when endA was mutated by transposon insertion, the nupG site was unintentionally mutated as well. There are no apparent effects of this mutation on cell function or growth.

References: 1) Nghiem, Y. et al. PNAS 85: 2709-2713. 2) Westh Hansen, S.V. et al. Eur. J. Biochem. 168: 385-391.

Are TOP10 cells lacIq+ (plus) or lacIq- (minus)? That is, do they produce the lambda lacIq repressor protein?

TOP10 cells are lacIq- (minus). They do not have the lacIq gene and therefore do not produce the lacIq repressor protein. lacIq is most commonly found on an F' episome, and therefore is present in TOP10F', JM101, JM109, and NM522 strains.

Why is Beta-mercaptoethanol (BME) no longer included with the One Shot Chemically Competent E. coli kits? What was the purpose of the BME during E. coli transformation?

Beta-mercaptoethanol (BME) degrades carbohydrates on the cell surface, which theoretically allows DNA to get closer to the membrane prior to heat shock. In the past, this was thought to improve the efficiency of transforming E. coli strains, and the addition of Beta-mercaptoethanol was a standard practice for all chemical transformations. However, galU and galK minus strains, such as TOP10, INV alpha F', DH5 alpha, DH10B and TOP10F', have fewer carbohydrates on the cell surface. After repeated testing of all of our strains, we determined that adding BME had no beneficial effect on transformation efficiency, and we chose to remove BME from the chemically competent One Shot kits.

Is the growth rate of TOP10 cells affected when harboring pZErO-1 plasmids?

Yes, the growth rate of TOP10 cells harboring pZErO-1 will be altered, and depends on how much functional ccdB protein is present. An insert fragment that does not completely disrupt the expression of the LacZ/ccdB fusion (usually very small inserts) will allow some production of the lethal protein which in turn will reduce the growth rate of the cell and produce lower plasmid yields. In contrast, a completely disrupted LacZ/ccdB fusion will allow normal (pUC level) growth and plasmid yield. Typical plasmid yields of pZErO-1 in LB/Zeocin media are 25% - 75% of pUC grown in LB/Amp. Colonies grown in SOB/Zeocin are more healthy and users can expect 75% - 200% of plasmid yield when compared to pUC grown in LB/Amp.

Are your E. coli strains derived from K12?

Most of our E. coli strains are K12-derived. The exceptions are the BL21 strains (derived from E. coli B), Mach1 (derived from E. coli W), and HB101. HB101 is derived from a K12/E. coli B hybrid - See FOCUS, 11:3, p. 56.

I found competent cell vials in my freezer with no box - how can I tell what strain/product it is?

Almost all Invitrogen competent cell vials are labeled by a laser with the strain name and a batch number. The label is etched into the plastic on the side of the vial, but it may be obscured from view by frost in the freezer.

The cap color can also be used to distinguish between products. Below is a list of cap colors for some of our products.

Chemically competent cells cap colors:
TOP10 One Shot - Purple; TOP10F' One Shot - Blue; One Shot Mach1 T1 Phage Resistant - Blue; One Shot OmniMAX2 T1 Phage Resistant - Pink; MAX Efficiency DH5? - Brown; Library Efficiency DH5? - Blue; Subcloning Efficiency DH5? - Clear; One Shot MAX Efficiency DH5?-T1 Phage Resistant - Yellow; One Shot DH10B T1 Phage Resistant - Green; INV?F' One Shot - Clear; MAX Efficiency Stbl2 - Green; One Shot Stbl3 - Clear; INV110 One Shot - Red; BL21 Star(DE3) - Red; BL21 Star(DE3)pLysS - Blue; BL21-AI - Orange; BL21(DE3)pLysE - Pink; BL21(DE3)pLysS - Green; BL21(DE3) - Brown

Electrocompetent cells cap colors:
TOP10 Electrocomp - Yellow; TOP10F' Electrocomp - Green; ElectroMAX DH10B - Yellow; ElectroMAX DH10B T1 Phage Resistant - Orange; ElectroMAX DH5?-E - Red; ElectroMAX Stbl4 - Clear

Does the methylation status of DNA affect its ability to be cloned?

Yes. Bacterial host cells will often degrade incoming DNA that has a methylation pattern that is "foreign" relative to that of the cell. Several host strains have been modified to accept mammalian methylation patterns. The modified markers include mcrA, mcrBC, and mrr. Also, endogenous (b-type) restriction endonucleases can be problematic. Modifications of the host to be rK- or rB- are necessary and include hsdR17(AK-, MK+), hsdR17(rK-, mK-), hsdS20(rB-, rB-) or hsdRMS. Strains with the hsdR17(rK-, mK+) mutation lack K-type restriction endonuclease, but contain K-type methylase. DNA prepared from hosts that are rK- mK- is unmethylated and will transform with lower efficiency in rK+ hosts.

TOP10, DH10B, and OmniMAX2-T1 cells contain the mcr, mrr, and hsdRMS mutations. Mach1 and standard DH5? strains only have the hsdR17(rK- mK+) mutation and are not recommended for cloning eukaryotic genomic DNA.

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.

How can AmpliTaq DNA Polymerase be inactivated after PCR?

There are several approaches that can be taken to inactivate the AmpliTaq DNA Polymerase after PCR.

(1) Because AmpliTaq DNA Polymerase is thermostable, it is necessary to heat it to high temperatures in order for it to be inactivated. Typically, a 99-100 degrees C for 10 min is sufficient.

(2) Raising the EDTA concentration to 10 mM will chelate any free Mg2+. Mg2+ is necessary for enzyme activity. By removing the Mg2+ the enzyme will no longer exhibit enzyme activity.

(3) Phenol-chloroform extraction of the PCR product and ethanol precipitation will also inactivate AmpliTaq DNA Polymerase.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.