the attB1 amp 2 sequences are relatively short and are therefore suitable for addition to genespecific primers After PCR amplification fragment contains the attB1 and attb2 sequences and can be used for recombination with a vector carrying the attP1 and atP2 sequences and the addition of th ID: 1043902
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1. B/P recombination of PCR fragment (example)the attB1 & 2 sequences are relatively short and are therefore suitable for addition to gene-specific primers After PCR amplification, fragment contains the attB1 and attb2 sequences, and can be used for recombination with a vector carrying the attP1 and atP2 sequences, and the addition of the BP clonase enzyme mix. The resulting Entry vector carries attL sequences and can be used for recombination into destination vectors with attR sequences.attB1 primer: 5´-GGGGACAAGTTTGTACAAAAAAGCAGGCT-(gene-specific sequence)-3´ attB2 primer: 5´-GGGGACCACTTTGTACAAGAAAGCTGGGT-(gene-specific sequence)-3´
2. B/P recombination of PCR fragment (example)
3. The subsequent movement of fragment into specialized destination vectorsOnce you have created an entry cloneYou can combine the entry vector with att L sites with a destination vector that has attR sequences and add LR Clonase mixtureThis results in recombination and vectors with attB and attP sequencesCan go in the opposite direction with BP clonase mixFigure from http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html
4. Selection for vectors that have undergone recombinationThe CcdB “cell death” geneThe CcdB protein is a potent poison of gyrase When an E. coli wild-type gyrA+ strain is transformed with a vector containing a functional ccdB gene, the gene product blocks bacterial growth. If ccdB is removed from vector by recombination before transformation, this recombinant plasmid no longer interferes with host viability. only cells containing recombinant plasmids give rise to colonies. This selection is an important part of the high success rate (90-95%) often experienced with the Gateway system. How to propagate vector containing the CcdB gene? gyrA462 mutation confers total resistance to CcdB Vectors can be amplified and prepared in large quantities in a gyrA462 host (DB3.1).
5. Web pages with examples of destination vectorsInvitrogen (search under “Gateway” Brand)http://www.invitrogen.com/Drosophilahttp://www.ciwemb.edu/labs/murphy/Gateway%20vectors.htmlPlantshttp://www.unizh.ch/botinst/Devo_Website/curtisvector/index_2.html
6. L1R1R3L3R2L2R4L4Multiple assembly all in one tube!! ccdB