Cloning procedure Three oligonucleotides are needed for the construction of the

Question

Cloning procedure

Three oligonucleotides are needed for the construction of the library:

ON-829: 5’- ACC ACC TCC GG-3’

ON-830: 5’- TTA CTT AGT TA-3’

LN (library of nucleotides): 5’- GA GGT GGT {NNK}n TAA CTA AGT AAA GC, where {NNK}n denotes a random region of the desired length ( 3 x n) and sequence.  NNK triplets specify random amino acids where N is one of the four nucleotides (A,C,G, or T), and K is either G or T.

Step 1: 5’-phosphorylated primers ON-829, ON-830, and the library of nucleotides (LN) are mixed together, heated at 70°C for 5 minutes, and slowly cooled down to 4°C.

Step 2: The SfiI-digested plasmid is purified away from the small fragment comprised between the two SfiI restriction sites.

Step 3: The purified and digested plasmid and the cold oligonucleotide mixture are combined and T4 DNA ligase is added.

Step 4: A mixture of dNTPs and a DNA polymerase are added to the ligation reaction.

Step 5: The plasmid DNA is then purified and used to transformed bacteria.

a) Represent, in a diagram, the structure of the assembled oligonucleotide complex obtained at the end of step 1. Write the sequence of each oligonucleotide (see above), label each oligonucleotide (including 5’ and 3’) and clearly show the double stranded regions inside the complex

Explanation / Answer

5'-------GA GGT GGT----{NNK}n---- TAA CTA AGT AAA GC-3'
3' GGC CT CCA CCA-5'-------------3' ATT GAT TCA TT-5'

each oligo is the reverse complement of one side of the library of nucleotides DNA sequence given, and so they will anneal to their complementary sequences...

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