1 The Gateway Cloning System Introduction to the Gateway System Contents
Defining Gateway technology.
Advantages of Gateway cloning.
Ways to enter the Gateway system.
How to obtain a Gateway expression clone.
2 The Gateway Cloning System
Maintains reading frame
No restriction enzymes
1 hour room-temperature reaction with gt99 efficiency
Compatible with automation
3 Key Benefits of the Gateway Technology
Efficiently and easily shuttle insert DNA from one expression plasmid to another
Simplify the cloning workflow and save time
Create expression clones without using restriction enzymes and ligase
Utilize Ultimate ORF clones a pre-made Gateway collection
Simultaneously clone in a specific order and orientation up to 4 DNA fragments into one plasmid
4 Phage lambda recombination in E. coli cos The Gateway System relies on five sets of specific and non cross-reacting att sequences l Phage attP 232 bp x attB E. coli 21 bp The specificity is given by the 7 nucleotides of the core region Integration (Int IHF) Excision (Int IHF Xis) attL attR Lysogen 96 bp 157 bp 5 Building a Gateway Entry Clone
BP Clonase II 6 Obtaining a Gateway Expression Clone
LR Clonase II 7 Gateway Cloning Platform a Proven Technology 800 700 600 500 Number of publications each year 400 300 200 100 0 1 2 3 4 5 6 Years after Gateway Technology was introduced
John M. Watson et al. FEBS Letters
The Gateway System seems to have become a universal standard in genomic research and there are many gene and gene fragment libraries in these Entry vectors.
8 Glossary of terms used in Gateway cloning att site A defined length of DNA that constitutes a recombination site. There are 4 classes of att sites called attB attP attL and attR. ccdB gene A counterselectable gene that allows for negative selection of unwanted by-product plasmids after recombination. Donor (pDONR) Vector A vector with attP sites flanking a counterselectable gene that recombines with a gene of interest flanked by attB sites. BP reaction A recombination event between attB and attP sites catalyzed by BP Clonase II Entry (pENTR) clone A vector that contains your gene of interest flanked by attL or attR sites. LR reaction A recombination event between attL and attR sites catalyzed by LR Clonase II Destination (DEST) Vector An application-geared vector with attR sites flanking a counterselectable gene that will recombine with one or more entry clones. MultiSite Gateway Technology A system that allows simultaneous assembly of multiple DNA fragments into a single destination vector 9 Policy for the use and distribution of Gateway Technology
The Gateway Technology is covered under Limited Use Label License. For more information regarding use please see LULL no. 19 at www.invitrogen.com.
Invitrogen offers distribution of Gateway entry clones as follows
Gateway entry clones containing attL1 and attL2 sites may be generated by academic and government researchers for the purpose of scientific research. Such clones may be distributed for scientific research by non-profit organizations and by for-profit organizations without royalty payment to Invitrogen.
Gateway expression clones containing attB1 and attB2 sites may be generated by academic and government researchers for the purpose of scientific research. Such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen. Organizations other than academic and government may also distribute such Gateway expression clones for a nominal fee payable to Invitrogen.
For inquiries please contact Invitrogens licensing department.
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