Molecular Biology Question Transformation. The table below (see next page) shows

Question

Molecular Biology Question

Transformation. The table below (see next page) shows approximate colony numbers (mock results) you might expect for some of the transformation treatments (See next laboratory session for more details of each condition). A couple of transformant colonies can be observed for the negative ligation control (transformation treatment II: pTAC-MAT-Tag-1 without any inserts), although none should be theoretically expected. Can you propose a molecular mechanism explaining how the pTAC-MAT-Tag-1 pre-digested with Hind III and EcoRI can be recircularized once in a while? Determine the competency of the competent bacteria used in this experiment (see Appendix D5).

Explanation / Answer

a) Restriction site for Hind III is A^AGCTT/TTCGA^A (^ refers to site of cleavage). Therefore, digestion with Hind III will produce AGCTT/A as sticky end. Restriction site for EcoR I is G^AATTC/ CTTAA^G  that will produce G/CTTAA as sticky end. So, if the DNA double digested with Hind III and EcoR1 circularize, it will result in a sequence GAGCTT/CTTAAA(sequence in bold is sticky end for Hind III). In this sequence i.e. GAXXTT/CTXXAA (X represents the bases that are not complementary to each other), 4 out of 6 bases are complementary to each other hence will base pair to form circular DNA molecule that is not too stable. If this happens, it will result in few colonies, as observed in case II.

b) Transformation efficiency (T.E) is defined as the number of colony forming units (cfu) that are produced by transforming 1 µg of plasmid into a given volume of competent cells.

Transformation efficiency (TE) is calculated using the following equation:
TE = no. of colonies X Dilution factor /amount of DNA (µg)
no of colonies = 88

µg of DNA=0.001

dilution factor =10 X

T.E =88* 10/0.001 = 8.8 *10 5 cfu/ µg

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