Stratagene pfu turbo pdf

Pfuturbo stratagene corporation samples were separated on a denaturing six percent acrylamide gel, 0. Qpcr applications using stratagenes mx realtime pcr. Pfuturbo cx hotstart dna polymerase boosts your bsseq. Pfuturbo and pfuultra are trademarks of stratagene. Approximately 25 pmol of biotinylated pcr product was captured on streptavidin. Pfuturbo dna polymerase is an enhanced version of pfu dna polymerase for robust, highfidelity pcr. Whether you are new to or experienced in qpcr, your individual needs are met with our comprehensive range of products and industryleading support. As with the original pfuturbo dna polymerase, pfuturbo dna polymerase ad is a mixture of cloned pfu dna polymerase and the exclusive thermostable archaemaxx pol ymeraseenhancing factor 1 that enhances pcr product yields and increases target length. Sitedirected mutagenesis is an invaluable tool to modify genes and study the structural and functional properties of a protein, based on the structure, function, catalytic mechanism, and catalytic residues of enzymes. Initially, gelpurified pcr products from step 1 were used for ligation step 2. Stratagene provides a total solution approach to realtime quantitative pcr qpcr, simplifying the challenges you face in sample preparation, assay setup, and data analysis. Introduction pfuturbo dna polymerase ad features a reformulated buffer system for increased economy with the same high performance as the original stratagene pfuturbo dna polymerase for robust, high.

A mutant pfu dna polymerase designed for advanced uracil. Us7704712b2 dna polymerase fusions and uses thereof. Sitedirected mutagenesis stratagene protocol mcmanus lab. The effects of different dna polymerases on the production of str stutter bands jim streeter montana dept. Pfu dna polymerase is an enzyme found in the hyperthermophilic archaeon pyrococcus furiosus, where it functions to copy the organisms dna during cell division. Genetic identification of a respiratory arsenate reductase. Sitedirected mutagenesis stratagene protocol this is the protocol for site. Pfuturbo dna polymerase is a mixture of cloned pfu dna polymerase and the exclusive thermostable archaemaxx polymeraseenhancing factor that enhances pcr product yields and increases target length capability without altering dna replication fidelity. Before assuming this will work for you, you will need to check the fermentas buffer.

Ah, but pfu turbo cx hotstart isnt your normal pfuits a mutated version thats been developed to fly through us without a second thought, so you wont have to. But this is with the stratagene pfu turbo, ultra, or ultraii. Quick test of the stratagene robocycle gradient 96 running through an abbreviated cycle at 10 seconds per block. Site directed mutagenesis an overview sciencedirect topics. Single worm pcr pcr was performed on single worms either to genotype c. The 12 kb pcr product was cloned and sequenced over the 2 kb assembly gap found in this region of scaffold 14979. A new dnabased test for detection of nucleophosmin exon. Pfu dna polymerase is used to ensure blunt ends and avoid secondsite mutations. In the laboratory setting, pfu is used to amplify dna in the polymerase chain reaction pcr, where the enzyme serves the central function of copying a new strand of dna during each extension step.

Moreover, pfu buffer was found to be compatible with ligase buffer. An enhanced version of pfu dna polymerase for robust, highfidelity pcr. Winter et al supplemental data journal of biological. Sitedirected mutagenesis method optimization ive done mutagenesis for 3 month, but until now all of my colonies have been only wild type. Supercompetent xl1blue ecoli stratagene was then transformed with the thus generated.

Subsequently, we found that pcr products could be directly used without removing pcr primers figure 2. Pcr reagents and instrumentation interactive catalog. We follow the protocol except use pfu turbo and 1 min per kb extension times. Stratagene scientists isolated the archaemaxx factor from pyrococcus furiosus and added it to our highfidelity enzymes, where it eliminates a pcr inhibitor and promotes shorter extension times, higher yield and greater target length.

Sitedirected mutagenesis includes single and combinational mutations. Primer selection try to achieve similar tm for all primers. Tm 4 kcalmol to avoid stable primer dimers design via software always use the same. Agilent technologies pfu turbo dna polymerase pfu turbo. Pfu turbo 10x pfu turbo buffer dntps 10mm forward and reverse primers 0.

We study the molecular mechanisms by which chromatinsignaling networks effect nuclear and epigenetic programs, and how dysregulation of these pathways leads to cancer and other human diseases. I think the stratagene buffer is proprietary, but dpni might tolerate a range of buffers. Pfu dna polymerase product information 9pim774 promega. As with the original pfuturbo dna polymerase, pfuturbo.

Using standard pfu polymerase conditions, we next compared the performance of the purified pfuv93qs7 enzyme termed pfux7 with the commercial pfuturbo pfut, stratagene, pfus7 phusion, the commercial pfuv93q turbo pfucx, stratagene, and pfuv93q using both standard and uracilcontaining primers, in a challenging mutagenesis reaction, on a. Pfuturbo dna polymerase is a mixture of cloned pfu dna polymerase and the exclusive thermostable archaemaxx polymeraseenhancing factor that enhances pcr product yields. Site directed mutagenesis this is the protocol for site directed mutagenesis based on the stratagene protocole. Pfu dna polymerase is recommended for use in pcr and primer extension reactions that require high fidelity 14. Mx3000p and mx3005p qpcr systems setup and users guide 5 4. Using standard pfu polymerase conditions, we next compared the performance of the purified pfuv93qs7 enzyme termed pfux7 with the commercial pfuturbo pfut, stratagene, pfus7 phusion, the commercial pfuv93q turbo pfucx, stratagene, and pfuv93q using both standard and uracilcontaining primers, in a. Agilent technologies building upon stratagenes legacy of excellence provides a. Our work centers on the biology of lysine methylation, a principal chromatinregulatory mechanism that directs epigenetic. Site directed mutagenesis with stratagene pfu turbo procedure.

It seems in your case that the anneal temps you use are going to be key since your tms will be very low. Us7704712b2 us10805,650 us80565004a us7704712b2 us 7704712 b2 us7704712 b2 us 7704712b2 us 80565004 a us80565004 a us 80565004a us 7704712 b2 us7704712 b2 us 7704712b2 authority us united states prior art keywords dna dna polymerase fusion polymerase pcr prior art date 20030325 legal status the legal status is an assumption and is not a legal conclusion. A complete list of distributors is available at page 3. Add 1 microliter pfu turbo polymerase stratagene incubate. Stratagene pfuturbo cx hotstart dna polymerase, is formulated with a. The archaemaxx factor is a key ingredient in many of stratagenes. Pfu turbo dna polymerase extends and incorporates the mutagenic primers, resulting in nicked circular strands. Stratagene robocycler gradient 96 run test youtube. Stratagene european contacts location telephone fax technical services. Dnapfu pyrococcus furiosus pcr stratagene archaemaxx 72 2 minkb dna 1 minkb 610 kb 1015 kb 2 minkb dmso. Our phusion dna polymerases are considered a gold standard for highfidelity pcr applications requiring high accuracy during dna amplification. The effects of different dna polymerases on the production. This is the protocol for sitedirected mutagenesis based on the stratagene kit. Future multiplexing or use of taqman assays in mind forward and reverse primer should have.

With the voltage labels facing out, insert the fuse holder into the fuse box so that the label for the desired voltage is in the proper orientation. Fusion pcr products with spei ends, underlined, generated by using pfu turbo stratagene, were prepared in two sequential pcrs that involved amplification of 1kb regions up and downstream of. Strategene is the most comprehensive tool i have seen, and takes the delivery of personalized medicine to a new level. Sitedirected mutagenesis using pfu dna polymerase and t4. The flat edge of the fuse holder has two different voltage labels 115v and 230v printed in opposite orientation to each other. Combine the following in a pcr tube, addingtaq last. Coverage of the most snps and pathways including folate, methionine, transulfuration, biopterin and histamine. Stratagene s quikchange sitedirected mutagenesis kit allows sitespecific mutation in virtually any doublestranded plasmid, thus eliminating the need for subcloning into mbased bacteriophage vectors and for ssdna rescue. I only used the pfu turbo from startagene for difficult mutagenesis i got clones but after sequencing mutation is absent, or promoter is deleted.