5 (1 pt) You wish to confirm orientation of your insert by restriction digest. I

Question

5 (1 pt) You wish to confirm orientation of your insert by restriction digest. In the diagram below the insert is shown in the forward orientation, and all single-cutter enzymes are indicated. The +1 of the vector (the nucleotide labeled as #1 on the vector sequence) is also included on the diagram. Vector size (without insert): 3 KB Site of insertion: 1KB Site of EcoRI cut: 2KB Insert size: 1KB Xba I cuts: 50bp from the 5' end of the insert B9111 cuts: 500bp from the 5' end of the insert Xba I Bgl 11 EcoRI Which enzyme or enzymes would you use for a diagnostic digest that will allow you to determine the orientation of your insert? a) b) predicted product sizes after digestion if insert is in forward orientation c) predicted product sizes after digestion if insert is in reverse orientation:

Explanation / Answer

Answer:

5. (a) EcoR1/Xba 1

(b) Forward direction:

EcoR1/Xba 1 would yield a digested product of (1 kb + 950 bp) = 1.95 kb

The other fragment would have a size of 2.05 kb.

(c) Reverse direction:

EcoR1/Xba 1 would yield a digested product of (1 kb + 950 bp) = 1.05 kb

The other fragment would be 2.95 kb.

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