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

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Answer

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® OmniMAX™2 T1 Phage Resistant - Pink; MAX Efficiency™ DH5α™ - Brown; Library Efficiency® DH5α™ - Blue; Subcloning Efficiency™ DH5α™ - Clear; One Shot® MAX Efficiency DH5α-T1™ Phage Resistant - Yellow; One Shot® DH10B™ T1 Phage Resistant - Green; INVαF' 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™ DH5α-E™ - Red; ElectroMAX™ Stbl4™ - Clear

Answer Id: E3345

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768355a64aa44ede9ba9a343949921de_FAQ

Are your E. coli strains derived from K12?

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Answer

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.

Answer Id: E3346

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1b03feb45062d109816cb3b00056e554_FAQ

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

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Answer

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.

Answer Id: E3356

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838b175bff4763ab697931f75d01bfd1_FAQ

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?

Product FAQ

Answer

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, INVαF', DH5α™, 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.

Answer Id: E3359

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2cacf394c64d587bff407ea47b9fd5e9_FAQ

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

Product FAQ

Answer

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.

Answer Id: E3361

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28c29a6e6e82ee4e9b0e537847200078_FAQ

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?

Product FAQ

Answer

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.

Answer Id: E3784

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91f83a19506cc93862726e116be9db12_FAQ

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

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Answer

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.

Answer Id: E3845

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63006cff9bec9dc6e8b0c7ce150a6aa6_FAQ

I am trying to clone an insert that is supposedly pretty toxic. I used DH5α and TOP10 cells for the transformation and got no colonies on the plate. Do you have any suggestions for me?

Product FAQ

Answer

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

- After transforming TOP10 or DH5α 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.

Answer Id: E7646

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3cc4791dca6824d082228a9a9bc66c9f_FAQ

What is the difference between TOP10 and TOP10F' cells?

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Answer

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.

Answer Id: E7661

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6c233779addbcbb5926683d7dc8fd489_FAQ