Gateway-Entry-Options-seminar

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Invitrogen Proprietary & Confidential,#,The Gateway Cloning System,How to generate an entry clone,Contents,Options for entering the Gateway,system,Defining the BP Clonase,reaction,Defining the LR Clonase,reaction,Description of Ultimate,ORF collection,Description of Vector NTI Advance,software for,in silico,cloning,Invitrogen Proprietary & Confidential,2,The Gateway Reactions,Invitrogen Proprietary & Confidential,3,Different ways to generate the entry clone,TOPO Cloning,TOPO,BP Clonase,BP Cloning,PCR Product,+,TOPO-Activated,Entry Vector,L1,L2,Gene,+,attB,PCR Product,B2,Gene,B1,Donor Vector,P2,ccdB,P1,Entry Clone,L2,Gene,L1,+,digested DNA Fragment,Gene,B1,digested Entry Vector,L2,L1,4. Pre-made entry clone,5. Custom-made entry clone,Ligase,Restriction/Ligase Cloning,ORF Collection,L2,ORF,L1,Invitrogen Proprietary & Confidential,4,BP Cloning The Reaction,90-99% correct clones,on Kan plates,+,+,BP Clonase,Invitrogen Proprietary & Confidential,5,BP Cloning - Primer Design for PCR,GGGG and the,att,B1 sequence must be added to the 5-primer (sense),GGGG and the,att,B2 sequence must be added to the 3-primer (antisense),att,B1,5 ,GGGG,ACAAGTTTGTACAAAAAAGCAGGCT,NNN,att,B2,5 ,GGGG,ACCACTTTGTACAAGAAAGCTGGGT,NNN,Gene Specific,Primer Sequence,Invitrogen Proprietary & Confidential,*After overnight incubation,BP Cloning Some Examples,Size (kb),PCR DNA,(fmol),PCR DNA,(ng),Colonies/,l,Transformation,Correct,Clones/Total,Clones Examined,0.26,15,38,3,7.5,1223,2815,10/10,1.0,15,38,10,25,507,1447,49/50,1.4,15,38,14,35,271,683,48/50,3.4,15,38,34,85,478,976,9/10,4.6,15,38,46,115,190,195,10/10,6.9,15,38,69,173,30 (235)*,54 (463)*,47/50,10.1,7.5,37.5,50.5,252.5,16 (112)*,42 (201)*,15/16,Tyrosine,Kinase,Transferrin,Receptor,Target Template:,EIF4e,b-Adaptin,MAP4,att,B-containing primers:,+ +,- -,+ +,- -,+ +,- -,+ +,- -,+ +,- -,Platinum,Pfx,DNA Polymerase,Platinum,Taq,DNA Polymerase High Fidelity,Platinum,Taq,DNA Polymerase,Total RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT RT,.,BP Cloning RT-PCR Using,att,B-Containing Primers,Invitrogen Proprietary & Confidential,8,TOPO Cloning -TOPOTA,Invitrogen Proprietary & Confidential,9,TOPO Cloning Directional TOPO,Invitrogen Proprietary & Confidential,10,Restriction/Ligase cloning,Use when there are convenient sites to cut insert out of another plasmid,Must cut out,ccdB,gene by using one of four RE sites flanking the ccdB,Reading frame of insert must be considered, as well as downstream expression elements,Various reading frames of pENTR vectors are available,Invitrogen Proprietary & Confidential,11,Pre-existing ORF collection,16,272 human ORFs (Oct 2006,release),Amber stop codons,Sequence verified,Ready to use in LR reactions, Ultimate ORF collection,Invitrogen Proprietary & Confidential,12,5. Custom Gene Synthesis,Quick and cost-effective,No PCR amplification necessary,100% accuracy (sequence verified),Optional codon optimization for expression,Invitrogen Proprietary & Confidential,13,In silico,cloning using Vector NTI Advance,TM,10.3,Primers for PCR reaction,Cloning Strategy,DNA of interest,Invitrogen Proprietary & Confidential,14,Gateway Summary,