Documents & Support

What competent cells do you suggest if I'm performing the following applications - routine cloning, protein expression, library construction, high-throughput cloning, cloning unstable DNA? Product FAQ

Answer

Routine cloning (http://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-applications/competent-cells-for-routine-cloning.html)
- Protein expression (http://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-applications/comp-cells-for-protein-expression.html)
- Library construction (http://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-applications/competent-cells-for-library-construction.html)
- High-throughput cloning (http://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-applications/competent-cells-for-high-throughput-cloning.html)
- Cloning unstable DNA (http://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-applications/competent-cells-for-cloning-unstable-dna.html)

Answer Id:: E6702

Was this answer helpful?

Yes No

Thank you for your response

Are the TA or TOPO TA Cloning vectors available to purchase without competent cells? What about your Zero Background, Zero Blunt, or Zero Blunt TOPO Cloning Kits Product FAQ

Answer

Most of our cloning vectors are offered in a complete format, which includes competent cells. While in most cases other cells can be used with our vectors, we cannot guarantee the results you will get with our cloning vectors if you use your own competent cells. For this reason, most TOPO TA Cloning vectors can only purchased in a complete kit with cells, although there are a few exceptions. Non-TOPO vectors are generally available in multiple formats, with and without cells. To get the most current information on available products, visit the Cloning section of our website under Products & Service.

Zero Background and Zero Blunt vectors are available without competent cells provided, but you should especially careful in choosing competent cells to use with them. These vectors contain the ccdB gene for efficient negative selection of clones without insert, and some E. coli strains are not compatible with the mechanism of negative selection by the lethal activity of the ccdB gene product. In particular, cells with the F' episome have a ccd locus containing the ccdA gene, which prevents ccdB protein cell-killing. Therefore, cells without the F' episome are recommended so that only the CcdB protein will be expressed, and its cell-killing ability will not be inhibited or reduced by CcdA.

Answer Id:: E3331

Was this answer helpful?

Yes No

Thank you for your response

Do any Invitrogen competent cells contain DMSO in the freezing medium? Product FAQ

Answer

Yes, several of our competent cells products are frozen with DMSO. The presence of DMSO (dimethylsulfoxide) will generally be indicated in the MSDS files if you have a question about a particular product, but here is a list of commonly used products that are known to have DMSO in the freezing buffer:

One Shot OmniMAX 2 T1 Phage Resistant Cells, Cat. No. C8540-03

One Shot INV?F' Chemically Competent Cells, Cat. No. C2020-03 and C2020-06

One Shot MAX Efficiency DH5?-T1 Chemically Competent Cells, Cat. No. 12297-016

MAX Efficiency DH5?-T1 Phage Resistant Cells, Cat. No. 12034-013

MAX Efficiency DH5? Chemically Competent Cells, Cat. No. 18258-012

Library Efficiency DH5? Chemically Competent Cells, Cat. No. 18263-012

MAX Efficiency DH5? F'IQ Cells, Cat. No. 18288-019

MAX Efficiency Stbl2Chemically Competent Cells, Cat. No. 10268-019

Answer Id:: E3355

Was this answer helpful?

Yes No

Thank you for your response

Can I re-use my competent cells once the tube has been thawed? Product FAQ

Answer

Yes, competent cells can be thawed and re-frozen at least once, but be aware that each freeze-thaw cycle can result in up to a 10-fold reduction in transformation efficiency.

To re-freeze unused competent cells, we recommend the following protocol: Pre-cool some new empty vials on ice for 5 min. Thaw the cells, and then aliquot a single-use volume of cells (usually 20-100 ul as recommended in the product manual) into the new tube. Freeze the cells immediately in a dry ice-ethanol bath. (Be sure that ethanol does not leak inside the tube - keep the level of ethanol well below the cap.) Transfer the frozen cells immediately to a -80C freezer, and do not thaw them again until ready for use.

Answer Id:: E3103

Was this answer helpful?

Yes No

Thank you for your response

What are some tips you can give me to obtain the highest transformation efficiency with my competent cells? Product FAQ

Answer

Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.

Answer Id:: E6709

Was this answer helpful?

Yes No

Thank you for your response

I accidentally ordered electrocompetent cells instead of chemically competent cells, but I don't have an electroporation apparatus. May I use the electrocompetent cells as chemically competent ones to do the heat shock transformation? Product FAQ

Answer

No, unfortunately not. Electrocompetent cells are not chemically treated. Unlike chemically competent cells, electrocompetent cells have an intact cell membrane.

Answer Id:: E6769

Was this answer helpful?

Yes No

Thank you for your response

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 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

Answer Id:: E3345

Was this answer helpful?

Yes No

Thank you for your response

Is it possible to perform a fast transformation protocol with the MultiShot competent cells? Product FAQ

Answer

There are situations where either because of time or process limitations, elimination of the heat shock and recovery time post-heat shock in the transformation protocol would be desirable. Testing was performed on many of our chemically competent cells in both the MultiShot StripWell and MultiShot FlexPlate format with a shortened transformation protocol. Use of a rapid transformation protocol (add DNA, no heat shock, no recovery period, plate on warm agar) yielded up to a log lower in transformation efficiency using the control DNA. This rapid transformation protocol can be performed with expectation of lower colony counts than our standard high efficiency transformation protocol and can only be performed with plasmids that use ampicillin as their antibiotic resistance. The rapid protocol is ideal when using a liquid handling system for high-throughput transformations with high cloning efficiency. Please see the product manual for full details on this rapid transformation protocol option.

Answer Id:: E16186

Was this answer helpful?

Yes No

Thank you for your response

I have a TOPO TA Cloning kit with TOP10 cells. I ran out of competent cells but still have vector left. I also have subcloning DH5? cells and TOP10F' cells in the freezer. Are either of these cells compatible? What strain features should I be aware of? Product FAQ

Answer

Subcloning DH5? cells are a compatible strain, but you will get lower efficiency (10e6 vs 10e9) and therefore risk getting fewer clones. Top10F' is also compatible, but if blue/white screening is performed, IPTG along with X-gal will be needed for detection due to the expression of the lacIq repressor present in cells containing an F' episome.

Answer Id:: E6734

Was this answer helpful?

Yes No

Thank you for your response

Can I use my own competent cells to transform my Invitrogen GeneArt CRISPR nuclease vector after ligation? Product FAQ

Answer

Yes, we provide kits both with or without competent cells. However, the vectors were optimized using Invitrogen One Shot TOP10 chemically competent E. coli, designed for high-efficiency cloning and plasmid propagation and stable replication of high-copy number plasmids. Please note that using competent cells of different genotype may lower cloning efficiency and can also result in a higher proportion of vectors without insert

Answer Id:: E10147

Was this answer helpful?

Yes No

Thank you for your response

My competent cells were stored in liquid nitrogen. Can I use them? Product FAQ

Answer

We do not recommend storage of competent cells in liquid nitrogen, as this will harm the cells. Additionally, the tubes that the competent cells were supplied in may not be able to withstand this temperature, leading to cracking or breaking.

Answer Id:: E6723

Was this answer helpful?

Yes No

Thank you for your response

What are some of the competent cell strains offered with your TA Cloning and TOPO TA Cloning vectors, and how should I choose? Product FAQ

Answer

TOP10, TOP10F', DH5a and INValphaF' are some of the strains offered with the Original TA Cloning Kits and the TOPO TA Cloning Kits. E. coli strains DH5a,TOP10, and INValphaF' do not have the lacIq repressor and permit constitutive expression from the lac promoter. With these cells, there is no need to add IPTG (inducer) for blue/white screening with X-gal. In contrast, E. coli strain TOP10F' carries the lacIq repressor and requires IPTG inducer to be added to enable blue/white screening. Please note, the F' episome in the INValphaF' strain is different from other strains in that it does not contain the lacIq repressor. Usually, presence of the F' and lacIq is only an advantage if you work with an insert that is potentially toxic to the host cell. If your insert is (potentially) toxic, we recommend using the TOP10F' cells without adding any IPTG. The lacIq repressor will repress expression from the lac promoter and you won't get blue-white screening, but you will still get colonies. This way you can clone a toxic construct.

TOPO TA cloning kits are also offered with Mach1 T1r competent cells. The Mach1 T1 Phage-Resistant (T1R) E. coli strain is the fastest growing chemically competent strain currently available. Doubling time is approximately 50 minutes, compared with an excess of 74 minutes for other cloning strains. Mach1 colonies are clearly visible within eight hours of plating the transformation mix, enabling you to plate and pick colonies in the same day. With these cells, there is no need to add IPTG (inducer) for blue/white screening.

Answer Id:: E3329

Was this answer helpful?

Yes No

Thank you for your response

What kind of competent cells should I use to propagate Donor vectors and Destination vectors? What cells should I use after the BP or LR recombination reaction? Product FAQ

Answer

All Donor vectors and Destination vectors contain the ccdB cell death gene to reduce background of non-recombined BP/LR plasmids. Therefore, growing non-recombined vector requires special cells (One Shot ccdB Survival 2 T1R Competent Cells) which are resistant to the lethal effects of ccdB. On the other hand, general E. coli cloning strains including TOP10 or DH5a may be used for plating the BP or LR reaction, or for propagation and maintenance of recombined Gateway constructs.

Answer Id:: E6804

Was this answer helpful?

Yes No

Thank you for your response

Can I use chemically competent cells with the CloneMiner II cDNA Library Construction Kit? Product FAQ

Answer

We do not suggest using chemically competent cells. There may be as much as a 100-fold reduction in the number of primary clones if chemically competent cells are used.

Answer Id:: E7343

Was this answer helpful?

Yes No

Thank you for your response

What is the storage media for the competent cells? Product FAQ

Answer

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.

Answer Id:: E6719

Was this answer helpful?

Yes No

Thank you for your response

Results per page
    spinner