UMass Amherst BIOCHEM 196 - 2013 BIOCHEM Project 1.doc (2 pages)

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Bio Chem September 26 2013 Project 1 Gene Recombination of GFP KanR Question 1 The restriction enzymes that we will be using to construct GFP Kanr plasmid are SACI and SA I In order to receive the GFP we must cut pBAG starting at base pair 1503 using enzyme SA I Then we will end GFP at base pair 657 enzyme SacI This way we will be extracting the GFP from pBAG leaving the Amp gene behind For pKan to receive GFP we must cut a section where the enzymes will match Starting at base pair 2484 using enzyme SA I then ending at base pair 2097 enzyme SACI this creates a space for GFP to enter Question 2 After cutting using the enzymes there will be 2 waist pieces pBAG without GFP section and from base pair 2484 to 2097 on pKanR Then we will combine the other 2 pieces to make GFP KanR inserting GFP base pair 1503 657 into pKan base pair section 2484 2097 for a total of 4 different pieces pieces Question 3 GFP strand 846 Base pairs pBAG plasmid without GFP 2878 Base pairs waste plasmid pKan waste strand 387 Base pairs pKan plasmid without waste strand 3820 Base pairs Question 4 GFP 846 base pairs pKan plasmid without waste strand 3820 base pairs 4666 base pairs with in the new GFP KanR plasmid Question 5 Original Cut Final Cut SACI A BAMHI A BAMHI B HINDIII B KPNI C SA I C GXHOI D Possible Combinations AA BA CA AB BB CB AC BC CC AD BD CD For a total of 12 possible ligates recombines plasmid pieces