Please READ QUESTION PROPERLY AND ANSWER FULLY! Q1. a) Assemble ICE-CREAM-SUNDAE
ID: 300866 • Letter: P
Question
Please READ QUESTION PROPERLY AND ANSWER FULLY!
Q1. a)
Assemble ICE-CREAM-SUNDAE by moving ICE first into CREAM and then resulting ICE-CREAM into SUNDAE to complete a plasmid containing ICE-CREAM-SUNDAE.
Use the iGEM standard assembly method (RFC 10)
Please answer the question fully and properly. Each step should have two sections
A) Describe what you would do
B) What would be produced (the fragments)
Explanation / Answer
A. step 1: For moving ICE, restriction enzyme Eco R1 and Spe1 will be used to to cut the ICE fragment. A gap will be created in CREAM by restriction enzyme EcoR1 and Xba1. Both the reaction product will be purifyied by gel electrophoresis. Insert of ICE and cut of CREAM will be ligated resulting in ICE-CREAM. flanking sites of Xba1 and Spe1 are same.
step 2: Now SUNDAE will be cut using Xba1 and Pst1, this insert will be moved into above created ICE-CREAM at 5' end. ICE-CREAM will be cut for makeing a gap for insertion of SUNDAE by Spe1 and Pst1. Desired fragments will be purified using gel electrophoresis. A ligation reaction of ICE-CREAM cut by Spe1 and Pst1 with insert of SUNDAE cut by Xba1 and Pst1 will resutl in the formation of ICE-CREAM-SUNDAE.
B. Fragments of step 1: ICE cut by EcoR1 and Spe1, a open plasmid having sticky end for EcoR1 and Xba1 or Spe1.
Fragments of step 2: SUNDAE cut by Xba1 and Pst1, and open plasmid created in step 1.
Q1. forword primer 3'- GAATTCGCGGCCG-5' Tm 61
reverse primet 3'- CTGCAGCGGCCC-5' Tm 64