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topota_man - User Manual TOPO TA Cloning ® Five-minute...

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Unformatted text preview: User Manual TOPO TA Cloning ® Five-minute cloning of Taq polymerase-amplified PCR products Catalog nos. K4500-01, K4500-40, K4510-20, K4520-01, K4520-40, K4550-01, K4550-40, K4560-01, K4560-40, K4500-02, K4510- 2 2 (pCR ® 2.1-TOPO ® ) Catalog nos. K4600-01, K4600-40, K4610-20, K4620-01, K4620-40, K4650-01, K4650-40, K4660-01, K4660-40 (pCR ® II-TOPO ® ) Version U 10 April 2006 25-0184 ii iii Table of Contents Kit Contents and Storage.................................................................................................................................... iv Accessory Products............................................................................................................................................. vii Methods ......................................................................................................................... 1 Overview.................................................................................................................................................................1 Producing PCR Products ......................................................................................................................................3 Setting Up the TOPO ® Cloning Reaction............................................................................................................4 General Guidelines for Transforming Competent Cells...................................................................................6 Transforming One Shot ® Mach1 ™-T1 R Competent Cells ..................................................................................7 Transforming One Shot ® DH5 α ™-T1 R , TOP10, and TOP10F´ Competent Cells ............................................9 Analyzing Transformants...................................................................................................................................12 Optimizing the TOPO ® Cloning Reaction ........................................................................................................14 Map of pCR ® 2.1-TOPO ® ......................................................................................................................................15 Map of pCR ® II-TOPO ® ........................................................................................................................................16 Performing the Control Reactions .....................................................................................................................17 Appendix...................................................................................................................... 20 Purifying PCR Products......................................................................................................................................20 Addition of 3´ A-Overhangs Post-Amplification ............................................................................................22 Recipes...................................................................................................................................................................23Recipes....
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This note was uploaded on 11/11/2011 for the course BIO 7.012 taught by Professor Lander during the Fall '10 term at MIT.

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topota_man - User Manual TOPO TA Cloning ® Five-minute...

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