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topota_man - TOPO TA Cloning Five-minute cloning of Taq...

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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
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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 ................................................................................................................................................................... 23 Technical Service .................................................................................................................................................. 24 Product Qualification .......................................................................................................................................... 25 Purchaser Notification ........................................................................................................................................ 26 References ............................................................................................................................................................. 27
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iv Kit Contents and Storage Shipping and Storage TOPO TA Cloning ® Kits are shipped on dry ice. Each kit contains a box with TOPO TA Cloning ® reagents (Box 1) and a box with One Shot ® Chemically Competent or Electrocomp cells (Box 2). TOPO TA Cloning ® Kits supplied with the PureLink Quick Plasmid Miniprep Kit (cat. nos.K4500-02 and K4510-02) are shipped with an additional box containing reagents for plasmid purification (Box 3). Store Box 1 at -20°C, Box 2 at -80°C, and Box 3 at room temperature. Types of TOPO TA Cloning ® Kits TOPO TA Cloning ® Kits are available with pCR ® 2.1-TOPO ® or pCR ® II-TOPO ® vector and a choice of One Shot ® Chemically or Electrocomp Competent cells as described in the table below. Select TOPO TA Cloning ® Kits are also available with PureLink Quick Plasmid Miniprep Kit. See page vi for the genotypes of the strains. Product Reactions One Shot ® Cells Type of Cells Catalog no. 20 TOP10 chem. competent K4500-01 40 TOP10 chem. competent K4500-40 20 Mach1 -T1 R chem. competent K4510-20 20 DH5 α -T1 R chem. competent K4520-01 40 DH5 α -T1 R chem. competent K4520-40 20 TOP10F´ chem. competent K4550-01 40 TOP10F´ chem. competent K4550-40 20 TOP10 electrocompetent K4560-01 TOPO TA Cloning ® Kit (with pCR ® 2.1-TOPO ® ) 40 TOP10 electrocompetent K4560-40 20 TOP10 chem. competent K4500-02 TOPO TA Cloning ® Kit (with pCR ® 2.1-TOPO ® and PureLink Quick Plasmid Miniprep Kit) 20 Mach1 -T1 R chem. competent K4510-02 20 TOP10 chem. competent K4600-01 40 TOP10 chem. competent K4600-40 20 Mach1 -T1 R chem. competent K4610-20 20 DH5 α -T1 R chem. competent K4620-01 40 DH5 α -T1 R chem. competent K4620-40 20 TOP10F´ chem. competent K4650-01 40 TOP10F´ chem. competent K4650-40 20 TOP10 electrocompetent K4660-01 TOPO TA Cloning ® Kit Dual Promoter (with pCR ® II-TOPO ® ) 40 TOP10 electrocompetent K4660-40 continued on next page
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v Kit Contents and Storage, continued TOPO TA Cloning ® Reagents TOPO TA Cloning ® reagents (Box 1) are listed below. Note that the user must supply Taq polymerase. Store Box 1 at -20°C.
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