Richard Lab:Amplified insert assembly: Difference between revisions

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Richard Lab:Amplified insert assembly (view source)

Revision as of 21:50, 18 March 2011

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The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids.  The eventual goal of assembly is to get these parts on the same plasmid next to one another.
The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids.  The eventual goal of assembly is to get these parts on the same plasmid next to one another.
==Materials==
===General===
*Pipettors
*Microcentrifuge
*Water baths
*Thermocycler
*Electroporator
*Selective media plates
===Enzymes===
*EcoRI Restriction Endonuclease
*XbaI Restriction Endonuclease
*SpeI Restriction Endonuclease
*PstI Restriction Endonuclease
*[http://www.neb.com/nebecomm/products/productR0176.asp DpnI] Restriction Endonuclease
*[http://www.neb.com/nebecomm/products/productM0254.asp Vent] DNA Polymerase (or another equivalent high fidelity polymerase)
*[http://www.neb.com/nebecomm/products/productM0289.asp Antarctic Phosphatase]
*T4 Ligase


==Procedure==
==Procedure==
Michael A. Speer
1,721

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