you have isolated the following linear fragment of cDNA Problem 1. You have isol

Question

you have isolated the following linear fragment of cDNA Problem 1. You have isolated the following linear fragment of cDNA from a jellyfish lambdaphage expression library. The cDNA sequence contains the luminous green fluorescent protein (GFP), green arrow Linear cDNA, length = 1.8 kb (1,800 base pairs) H3 Hind III H3 H3 R1 K H3 K K-Kpnl Coordinates are given in kilobases (kb) 0.1 0.5 0.8 1. 1.5 Green Fluorescent Protein (GFP) 1.0 Part A. You perform several restriction digests. List the number of DNA fragments (pieces) generated by a complete digestion with the following restriction endonucleases Digest # of fragments r n b EcoR I Hind III EcoR I & Kpn I Which of these restriction digests would be appropriate for cloning the GFP gene? Would your cloning strategy be directional or non-directional?

Explanation / Answer

Part A

a.) EcoR1 - No.of fragments: 2, Size of the fragments: 0.8 kb, 1 kb.

b.) Kpn1 - No.of fragments: 3, Size of the fragments: 1 kb, 0.5 kb, 0.3 kb.

c.) Hind 3 - No.of fragments: 4, Size of the fragments: 0.1 kb, 0.4 kB, 0.7 kB, 0.6 kB.

d.) EcoR1 and Kpn1 - No.of fragments: 4, Size of the fragments: 0.8 kB, 0.2 kB, 0.5 kB, 0.3 kB.

Part B: Hind3 would be appropriate for cloning the Gfp gene. This enzyme doesn't have cleavage site within the Gfp gene.

Part C: cloning strategy would be non directional because both ends of the Gfp gene is cleaved by the same enzyme hind3 so non-directional strategy has be followed.

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