Navigation Links
Enhanced PCR Cloning System


PCR-Script cloning kits feature new additions

Tim Sanchez Julie Robinson Mary Buchanan
Stratagene

The PCR-Script cloning kits are convenient for cloning PCR fragments generated with any PCR enzyme.1,2 The method is highly efficient, easy, and superior to other available methods.3 Now, Stratagene improves the kits by including both the StrataPrep PCR purification kit, so preparing PCR products is simple, and Epicurian Coli XL10-Gold ultracompetent cells,* so the highest transformation efficiencies of ligated DNA are attained. Furthermore, these innovative additions are available at no extra cost.

The core of these PCR-Script cloning kits is the PCR-Script cloning method,1 in which PCR products are incubated for a 1-hour ligation reaction with one of the Srf I-digested PCR-Script vectors, Srf I restriction enzyme, and T4 DNA ligase. The Srf I enzyme recognizes the rare oligonucleotide sequence 5-GCCCGGGC-3. By including the Srf I enzyme in the ligation reaction, the PCR-Script cloning method maintains high concentrations of digested vector DNA and allows the use of nonphosphorylated, unmodified PCR products. This method ensures a low background of recircularized vector and increased ligation efficiency for blunt-ended DNA fragments by the simultaneous, opposing reactivity of the Srf I enzyme and T4 DNA ligase on nonrecombinant vector DNA. Ligated DNA is then ready to be transformed into competent cells.

The PCR-Script kits, although ideal to use with PCR enzymes such as Pfu and pfuturbo DNA polymerases,,** are versatile and can be adapted for use with any PCR enzyme. Pfu and PfuTurbo DNA polymerases are proofreading enzymes that possess 3- to 5-exonuclease activity and generate high-fidelity, blunt-end PCR products.4,5 In cases where PCR products are generated with Taq DNA polymerase or other low-fidelity DNA polymerases, the PCR-Script kits include the reagents necessary to blunt the PCR products for cloning into the PCR-Script vectors.

From purification and cloning of PCR products to transformation of ligated DNA, Stratagene has optimized each step in the PCR-Script cloning kits by incorporating two new products: The StrataPrep PCR purification kit6 makes it easy to prepare PCR products, and XL10-Gold ultracompetent cells7 provide the highest transformation efficiencies of ligated DNA.

Purification of PCR Products

Following PCR amplification, cloning the PCR product requires an ethanol precipitation step to remove excess primers. When multiple samples are processed simultaneously, ethanol precipitation can be cumbersome. To simplify this procedure, all PCR-Script kits now include the StrataPrep PCR purification kit. StrataPrep kits are based on a special DNA-binding fiber matrix in a microspin format,6 which replaces ethanol precipitation of PCR products and allows quick, simultaneous processing of multiple samples. The StrataPrep PCR purification kit removes primers by centrifugal washing; the desired product is eluted in a small volume of buffer or water.

Figure 1

We compared the purity of 0.6- and 1.5-kb PCR products that were prepared using either the StrataPrep kit or the silica-gel-membrane method (Figure 1). Material stained with ethidium bromide and smaller than 100 bp was eliminated from 0.6- and 1.5-kb PCR products purified using the StrataPrep kit (lanes 3 and 7, respectively); whereas, the silica-gel membrane retained contaminants migrating between 72 and 118 bp (lanes 4 and 8, respectively). In another series of experiments, the PCR-Script cloning method yielded similar efficiencies and number of transformants, consistently producing greater than an 80% cloning efficiency for PCR fragments prepared by either ethanol precipitation or the StrataPrep kit (data not shown).

Highest Transformation Efficiencies of Ligated DNA

All PCR-Script kits were further improved by adding XL10-Gold Kan ultracompetent cells, which were created by Stratagene to improve the transformation efficiency of ligated DNA into E. coli.7 Ligated DNA molecules transform competent E. coli at a significantly lower efficiency than supercoiled molecules, and large plasmids transform cells less efficiently than small plasmids. This new E. coli host exhibits notably higher transformation efficiencies for ligated DNA and a larger colony size after transformation into XL10-Gold Kan cells; hence, colonies are scored and picked more quickly. Because the blue-white color selection in XL10-Gold Kan cells is unusually vivid, picking the conspicuous colonies is simple.

We tested the transformation efficiencies of the test insert ligated into the pPCR-Script vector into either XL10-Gold Kan or XL1-Blue MRF Kan cells. Four transformations into each of the two cell lines were performed in triplicate. These data confirm that XL10-Gold ultracompetent cells increase transformation efficiencies at least 10-fold over XL1-Blue cells (Figure 2).

Figure 2

Conclusions

Stratagenes PCR-Script cloning kits are outstanding for fast cloning of blunt-end PCR products, no matter what PCR enzyme is used to generate the inserts. Stratagene improved the kits to include the StrataPrep PCR purification kit and XL10-Gold ultracompetent cells. The StrataPrep kit allows PCR products to be rapidly and efficiently purified from excess primers. XL10-Gold Kan ultracompetent cells provide the highest transformation efficiency of ligated DNA and easy-to-score colonies. Finally, these improvements were added to the PCR-Script kit without increasing the price of the kit.

REFERENCES
  1. Bauer, J., et al. (1992) Strategies 5: 62-65.

  2. Pearson, S. and Bauer, J.C. (1996) Strategies 9: 26-27.

  3. Sanchez, T., Zheng, C.-F., and Bauer, J. (1996) Strategies 9: 44-46.

  4. Scott, B., et al. (1994) Strategies 7: 6263.

  5. Hogrefe, H., et al (1997) Strategies 10: 93-96.

  6. Braman, J. and Basehore, S. (1997) Strategies 10: 84-86.

  7. Jerpseth, B., Callahan, M., and Greener, A. (1997) Strategies 10: 37-38.

* U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending.
** U.S. Patent No. 5,545,552 and patents pending.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
2. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
3. Enhanced siRNA Delivery and Long-term Gene Silencing
4. UV Nicking of DNA in Agarose Gels for Enhanced Transfer and Detection of Megabase Size DNAs
5. Measuring Intracellular Enhanced Green Fluorescent Protein With the VersaFluor Fluorometer
6. Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein
7. Increased Analyte Sensitivity through the Utility of Enhanced Mass-Resolution on the FinniganTSQ Quantum Discovery
8. Novel Bonding Chemistry Imparts Enhanced Polar Selectivity to TSK-GEL ODS-100V Reversed Phase Columns
9. New Yeast Cloning System for Producing Proteins with Native Amino Acid Sequences
10. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
11. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Enhanced PCR Cloning System

(Date:8/28/2014)... converters are important devices in spintronics, an electronic ... of electrons but also on their spin and ... of electric into magnetic signals and vice versa. ... from the Institute of Physics at Johannes Gutenberg ... UK, Prague, and Japan, has for the first ...
(Date:8/28/2014)... 2014  Next month, executives from clinical trial marketing firm ... beginning with Patient-Centered Clinical Trials 2014 , to be ... , September 4-5. Patient recruitment experts Bonnie A. ... will share insights on the benefits of employing an ... – from media to mobile apps – can be used ...
(Date:8/28/2014)... 2014  Armetheon, Inc. ( www.armetheon.com ), a ... late-stage cardiovascular drug candidates, today announced that it ... of financing. The Series A round was co-led ... participation from investors that included Atheneos Capital and ... Larry Hsu , the founder of Impax Laboratories. ...
(Date:8/28/2014)... A new market research report ... available from Strategic Consulting, Inc., the leading information ... Market Review, Fourth Edition: Global Review of Microbiology ... and compares microbiology test volumes, market values and ... Asia, and forecasts future volumes and market values ...
Breaking Biology Technology:A new, tunable device for spintronics 2A new, tunable device for spintronics 3BBK Worldwide Leads Sessions at Key September Events 2Armetheon Completes $7.0 Million Series A Financing 2Armetheon Completes $7.0 Million Series A Financing 32 Billion Industrial Microbiology Tests Conducted for Product Quality and Safety Worldwide 22 Billion Industrial Microbiology Tests Conducted for Product Quality and Safety Worldwide 3
... years, I have to admit that Ive never been there. ... Lever Bros. (now Unilever ) in New York City during ... young, Indian man with a doctorate and other degrees from the ... he had just arrived in New York without any contacts. He ...
... incredibly bullish , ,Plymouth, Wis. - After years ... like Rust Belt, Wisconsin got a much needed boost from ... 8, 2003 edition. In an interview with Jeff Gendell, a ... that the U.S. is about to enter the strongest recovery ...
... is a national leader in the total number of ... a significant catalyst for new job creation as well, ... University of Louisville who recently addressed an audience at ... lecture series at the MGE Innovation Center , ...
Cached Biology Technology:Midwest Biotech: Watch India, the Next Biotech Frontier 2Midwest Biotech: Watch India, the Next Biotech Frontier 3Midwest Biotech: Watch India, the Next Biotech Frontier 4Midwest Biotech: Watch India, the Next Biotech Frontier 5Wisconsin Manufacturing Poised For a Barrons Bounce 2Wisconsin Manufacturing Poised For a Barrons Bounce 3Business Incubators Grow Economy and Jobs 2Business Incubators Grow Economy and Jobs 3
(Date:8/28/2014)... alert the world to Global Warming, has called for ... underground. He says that Carbon Capture, combined with limits ... avoid global warming getting out of control over the ... made the call during his presentation to the International ... meeting to discuss Carbon Capture and Storage. , ...
(Date:8/28/2014)... (LMU) in Munich have assigned a number of ... arthropods. These animals lived in shallow marine habitats ... found in Jurassic strata. , Before they ... habitat and were fossilized some 435 million years ... the Silurian seas although they were not ...
(Date:8/27/2014)...  WaferGen Bio-systems, Inc. (NASDAQ: WGBS) today announced that ... of units of common stock and warrants at a ... $20 million, prior to deducting underwriting discounts and commissions ... and warrants are immediately separable and will be issued ... offering consists of 2,000 shares of common stock and ...
Breaking Biology News(10 mins):Global warming pioneer calls for CO2 to be taken from atmosphere and stored underground 2Paleontology: Oldest representative of a weird arthropod group 2WaferGen Bio-Systems Announces Successful Completion of Public Offering Raising $20 Million 2WaferGen Bio-Systems Announces Successful Completion of Public Offering Raising $20 Million 3
... invites all journalists to attend its 64th Annual Scientific ... field of aging from November 18 to 22 ... charge. An estimated 3,500 professionals are expected to ... Veterans Memorial Convention Center and Sheraton Boston Hotel. The ...
... Later this week, the American Society of Plant Biologists (ASPB) ... to chart the future of the field. The Plant ... plant science research community in a process that will develop ... over the next decade. The summit will bring together ...
... researchers could help develop culturally appropriate conservation efforts and ... southwest China where deforestation is a major environmental issue. ... Religion and the Cultural Studies program, and An Jing, ... Studies, found a distinct link between the strong religious ...
Cached Biology News:Join GSA in Boston for the nation's premier aging conference 2ASPB convenes leaders to chart future of plant science research 2Religion and ecology among China's Blang people 2
Recognizes the ~115 kDa NMDA receptor NR1 subunit in rat cortex tissue extracts....
... formation of phosphodiester bonds in the presence ... hydroxyl and 5' phosphate termini. The unique ... can be performed in 5 minutes (1). ... this enzyme. A T4 DNA Ligase ...
Porcine Coronary Artery Smooth Muscle Cells (PCASMC) (>500,000 cells)...
Sulfadimethoxine (CH-2027)...
Biology Products: