Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Product FAQ

Answer

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.

Answer Id: E3098

Was this answer helpful?

Yes
No
7b9af43130a405a9d9e93366e5befc2c_FAQ

Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?

Product FAQ

Answer

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.

Answer Id: E3100

Was this answer helpful?

Yes
No
7823454388724302e4b98f8e4937b70f_FAQ

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

Product FAQ

Answer

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 OmniMAX™2-T1 cells contain the mcr, mrr, and hsdRMS mutations. Mach1 and standard DH5α™ strains only have the hsdR17(rK- mK+) mutation and are not recommended for cloning eukaryotic genomic DNA.

Answer Id: E3102

Was this answer helpful?

Yes
No
378a592620f9aa1fdb1d7f40611ba6e9_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

Was this answer helpful?

Yes
No
91f83a19506cc93862726e116be9db12_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
63006cff9bec9dc6e8b0c7ce150a6aa6_FAQ

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

Product FAQ

Answer

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.

Answer Id: E3860

Was this answer helpful?

Yes
No
74e0985805518e2e74bc2c97284b5962_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
768355a64aa44ede9ba9a343949921de_FAQ

Are your E. coli strains derived from K12?

Product FAQ

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

Was this answer helpful?

Yes
No
1b03feb45062d109816cb3b00056e554_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
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

Was this answer helpful?

Yes
No
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

Was this answer helpful?

Yes
No
28c29a6e6e82ee4e9b0e537847200078_FAQ

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

Product FAQ

Answer

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.

Answer Id: E3362

Was this answer helpful?

Yes
No
3a8be08c8d01829668b7dc8dd13c956e_FAQ

Why doesn't the 433A manual or the "quick start card" mention the need for an extra AA cartridge in the guideway at the start of a sequence?

Product FAQ

Answer

The newest 433A User's Manual does cover this. The barcode reader is one position ahead (left) of the needle position. The extra (empty) is needed to prevent advancement of the first cartridge until after it is read.

Answer Id: E1314

Was this answer helpful?

Yes
No
3f1503354f2db909967f808daddcfab3_FAQ

How can peptide synthesis amino acid cartridges leak and spill solvents during their activation and transfer to the RV?

Product FAQ

Answer

If these cartridges are being reused, the NMP can cause them to swell, and they no longer fit or slide well in the guideway. If the guideway or the exterior of the needles have became dirty, this can also lead to misalignment. And if you forgot to remove the metal cap, the needle cannot penetrate the septum - this may cause a spill OR stop the run.

Answer Id: E1316

Was this answer helpful?

Yes
No
2f0fb91467ba67924b591da3bec23914_FAQ

How can AmpliTaq® DNA Polymerase be inactivated after PCR?

Product FAQ

Answer

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.

Answer Id: E1319

Was this answer helpful?

Yes
No
c7919624bf79c38d54c892bfa5bca4c3_FAQ