Navigation Links
Highest Possible Transformation Efficiencies for High-Throughput ,,, Applications



Convenient, time-saving 96Pack Gold competent cells streamline transformation procedures

Julie Bonnardel Ronda Allen Bruce Jerpseth Sean Banks

Stratagene

The new 96Pack Gold competent cells* from Stratagene feature a convenient, high-throughput format that saves time without compromising quality. These cells feature exceptionally high transformation efficiencies, fast growth, blue-white color screening of recombinants, and high-quality plasmid miniprep DNA. Simplified transformations, fast results, and ease of use make these cells ideal for all high-throughput cloning applications. For transformations of supercoiled or ligated DNA, we compared the performance of 96Pack Gold cells with a competitors 96-well-format competent cells and found that our cells consistently yielded higher transformation efficiencies.

With continuing advances in molecular biology technology, high-throughput methods have become increasingly important. With the 96Pack Gold competent cells, Stratagene has combined convenience with superior quality to ensure the best possible performance in a high-throughput format.

Combining Highest Efficiency with a High-Throughput Format

Stratagenes Hte phenotype, introduced in 1997, represented a breakthrough in competent-cell technology. Until that time, the best possible transformation efficiency was just higher than 5 x 109 transformants per microgram of supercoiled pUC18 vector (2.7 kb); efficiencies for ligated DNA were lower as expected. With the advent of the Hte phenotype, transformation efficiencies for ligated DNA increased 25-fold. This phenotype allows competent cells to better accept ligated or larger DNA plasmids, which are known to transform less efficiently than small plasmids on a per molecule basis. Due to exceptional performance, we have found that competent cells with the Hte phenotype are the best choice for any cloning experiment that demands a high efficiency of retrieving transformants from ligation reactions, such as constructing plasmid libraries and PCR cloning. The Hte phenotype helps 96Pack Gold cells fulfill the need for streamlined transformation procedures and superior performance.

In the basic high-throughput procedure, DNA is added to each well of 96Pack Gold cells with a multichannel pipettor. The microtiter plate is chilled and then subjected to heat shock, which is simply performed with either a thermocycler, a heating block, or a conventional water bath. Growth medium is added to the wells. After incubations, transformants are selected with the appropriate antibiotic medium. This simple protocol saves time and reagents, while providing the highest possible transformation efficiencies.

Engineered to Achieve the Highest Performance

96Pack Gold competent cells carry the Hte mutation, which confers the capacity to transform large and ligated DNA molecules at a higher efficiency. The 96Pack Gold strain is endonuclease-deficient (endA1), which greatly improves the quality of plasmid miniprep DNA. These cells are recombination-deficient (recA), ensuring the stability of inserted DNA. With the lacIqZDM15 gene on the F episome, 96Pack Gold competent cells allow bluewhite color screening of recombinant plasmids. In addition, this cell strain is restriction-minus, a feature that increases the efficiency of introducing eukaryotic DNA into E. coli and increases the size and representation of libraries constructed with methylated or hemimethylated DNA. Finally, 96Pack Gold cells grow at a fast rate, resulting in the formation of larger, easier-to-select colonies (Figure 1).

Superior Performance

We compared the transformation efficiency of 96Pack Gold cells with MultiShot cells (Invitrogen) (Figure 2). To test efficiency with supercoiled DNA, we added 1 pg and 10 pg of the pUC plasmid to either 96Pack Gold or MultiShot cells. To test efficiency for ligated DNA, we transformed 96Pack Gold cells and MultiShot cells with 2.3 ng of a ligated construct, the pCMV-Script plasmid with the LacZ gene insert (approximately 4.9 kb). For each experiment, we followed the manufacturers protocols. As can be seen, 96Pack Gold competent cell transformation efficiency outperformed the competitors product with both supercoiled and ligated DNA.

Conclusions

Stratagenes high-efficiency 96Pack Gold competent cells, provided in a high-throughput format, are the superior choice for PCR cloning, restriction enzyme cloning, and ligase-independent cloning. In comparison transformations of either s upercoiled or ligated DNA, 96Pack Gold chemically competent cells yielded higher efficiencies than another 96-well-format product. We confirmed that these cells streamline transformation procedures, and outperform other commercially available cells.

REFERENCE

1. Jerpseth, B., et al. (1997) Strategies 10: 37-38.

Genotype

XL10-Gold strain: Tetr D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F proAB lacI qZDM15 Tn10 (Tetr) Amy Camr]

* U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell Line
2. New Competent Cells for Highest Transformation Efficiencies
3. Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System
4. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
5. Transformation of Nematodes With the Helios Gene Gun
6. Transformation of Filamentous Fungi by Microprojectile Bombardment
7. Transformation of Filamentous Fungi by High-Voltage Electroporation
8. High Electrotransformation Efficiencies Obtained With DNA From Ligation Mixtures
9. Innovative Tissue Array Technology for High-Throughput Screening of Gene Expression
10. High-Throughput Cultivation of Bacterial Clones
11. High-Throughput Isolation of Total RNA
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/11/2017)... ... October 11, 2017 , ... ... has granted orphan drug designation to SBT-100, its novel anti-STAT3 (Signal Transducer and ... of osteosarcoma. SBT-100 is able to cross the cell membrane and bind intracellular ...
(Date:10/10/2017)... , ... October 10, 2017 , ... ... Center’s FirstHand program has won a US2020 STEM Mentoring Award. Representatives of the ... for Excellence in Volunteer Experience from US2020. , US2020’s mission is to change ...
(Date:10/10/2017)... SANTA CRUZ, Calif. , Oct. 10, 2017 /PRNewswire/ ... SBIR grant from the NIH to develop RealSeq®-SC (Single ... preparation kit for profiling small RNAs (including microRNAs) from ... Cell Analysis Program highlights the need to accelerate development ... "New techniques for ...
(Date:10/9/2017)... , Oct. 9, 2017  BioTech Holdings ... mechanism by which its ProCell stem cell therapy ... limb ischemia.  The Company, demonstrated that treatment with ... of limbs saved as compared to standard bone ... molecule HGF resulted in reduction of therapeutic effect.  ...
Breaking Biology Technology:
(Date:4/13/2017)... , April 13, 2017 According to a new ... Authentication, Identity Analytics, Identity Administration, and Authorization), Service, Authentication Type, Deployment Mode, ... IAM Market is expected to grow from USD 14.30 Billion in 2017 ... (CAGR) of 17.3%. ... MarketsandMarkets Logo ...
(Date:4/11/2017)... , April 11, 2017 No two ... researchers at the New York University Tandon School ... Engineering have found that partial similarities between prints ... used in mobile phones and other electronic devices ... The vulnerability lies in the fact that ...
(Date:4/5/2017)...  The Allen Institute for Cell Science today announces ... portal and dynamic digital window into the human cell. ... application of deep learning to create predictive models of ... a growing suite of powerful tools. The Allen Cell ... publicly available resources created and shared by the Allen ...
Breaking Biology News(10 mins):