The Gateway® Cloning System

1. How to generate an entry clone The Gateway® Cloning System 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

2. The Gateway® Reactions Invitrogen Proprietary & Confidential

3. L1 L2 L1 L2 + B1 Gene + Gene digested Entry Vector TOPO-Activated Entry Vector digested DNA Fragment PCR Product P1 P2 ccdB + B1 Gene B2 Donor Vector PCR Product attB Different ways to generate the entry clone BP Cloning TOPO® Cloning BP Clonase™ TOPO® Gene L1 L2 Entry Clone Ligase 4. Pre-made entry clone 5. Custom-made entry clone Restriction/Ligase Cloning L1 L2 ORF ORF Collection Invitrogen Proprietary & Confidential

4. 90-99% correct clones on Kan plates BP Cloning – The Reaction + + BP Clonase™ Invitrogen Proprietary & Confidential

5. Gene Specific Primer Sequence BP Cloning - Primer Design for PCR • GGGG and the attB1 sequence must be added to the 5’-primer (sense) • GGGG and the attB2 sequence must be added to the 3’-primer (antisense) attB1 5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN… attB2 5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN… Invitrogen Proprietary & Confidential

6. BP Cloning – Some Examples Correct PCR DNA PCR DNA Colonies/l Size (kb) Clones/Total (fmol) (ng) Transformation Clones Examined 15 3 1223 0.26 10/10 38 7.5 2815 15 10 507 1.0 49/50 38 25 1447 15 14 271 1.4 48/50 38 35 683 15 34 478 3.4 9/10 38 85 976 15 46 190 4.6 10/10 38 115 195 15 69 30 (235)* 6.9 47/50 38 173 54 (463)* 7.5 50.5 16 (112)* 10.1 15/16 37.5 252.5 42 (201)* *After overnight incubation Invitrogen Proprietary & Confidential

7. BP Cloning – RT-PCR Using attB-Containing Primers Tyrosine Kinase Transferrin Receptor Target Template: EIF4e b-Adaptin MAP4 + + - - + + - - + + - - + + - - + + - - attB-containing primers: Platinum® Taq DNA Polymerase Platinum® Taq DNA Polymerase High Fidelity Platinum® Pfx DNA Polymerase Total RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT™ RT.

8. TOPO® Cloning -TOPO®TA 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 Invitrogen’s Ultimate™ ORF collection 16,272 human ORFs (Oct 2006 release) Amber stop codons Sequence verified Ready to use in LR reactions http://orf.invitrogen.com/cgi-bin/ORF_Browser 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 AdvanceTM 10.3 DNA of interest Primers for PCR reaction Cloning Strategy Invitrogen Proprietary & Confidential

14. Gateway® Summary Invitrogen Proprietary & Confidential