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The Gateway® Cloning System How to generate an entry clone Contents Options for entering the Gateway ® system Defining the BP Clonase reaction Defining.

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Presentation on theme: "The Gateway® Cloning System How to generate an entry clone Contents Options for entering the Gateway ® system Defining the BP Clonase reaction Defining."— Presentation transcript:

1 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

2 Invitrogen Proprietary & Confidential 2 The Gateway® Reactions

3 Invitrogen Proprietary & Confidential 3 Different ways to generate the entry clone 2.TOPO® Cloning TOPO®BP Clonase 1.BP Cloning PCR Product + TOPO-Activated Entry Vector L1L2 Gene + attB PCR Product B2GeneB1 Donor Vector P2 ccdB P1 Entry Clone L2 Gene L1 + digested DNA Fragment GeneB1 digested Entry Vector L2L1 4. Pre-made entry clone 5. Custom-made entry clone Ligase 3.Restriction/Ligase Cloning ORF Collection L2 ORF L1

4 Invitrogen Proprietary & Confidential 4 1.BP Cloning – The Reaction 90-99% correct clones on Kan plates ++ BP Clonase

5 Invitrogen Proprietary & Confidential 5 1.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… Gene Specific Primer Sequence

6 Invitrogen Proprietary & Confidential *After overnight incubation 1.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

7 Tyrosine Kinase Transferrin Receptor Target Template:EIF4eb-AdaptinMAP4 attB-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. 1.BP Cloning – RT-PCR Using attB-Containing Primers

8 Invitrogen Proprietary & Confidential 8 2.TOPO® Cloning -TOPO®TA

9 Invitrogen Proprietary & Confidential 9 2.TOPO® Cloning – Directional TOPO®

10 Invitrogen Proprietary & Confidential 10 3.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

11 Invitrogen Proprietary & Confidential 11 4.Pre-existing 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 Invitrogens Ultimate ORF collection

12 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

13 Invitrogen Proprietary & Confidential 13 In silico cloning using Vector NTI Advance TM 10.3 Primers for PCR reaction Cloning Strategy DNA of interest

14 Invitrogen Proprietary & Confidential 14 Gateway® Summary


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