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#EcoRI

3 public questions tagged with this topic.

Restriction Digestion & Mapping

EcoRI and BamHI cut linear DNA with 2 kb, 3 kb, 1 kb fragments. Label appears only on 2kb. Why?

When linear DNA is cut by two enzymes producing three fragments, each fragment has two ends derived from cleavage events. Ends originating from natural termini or restriction cuts may be blunt or sticky depending on enzyme specificity. If only 2 kb fragment becomes labeled after end-filling with Klenow and radiolabeled dNTPs, it must be the only fragment possessing a 5' overhang with recessed 3' OH that polymerase can extend using template strand. Other fragments bearing blunt ends or 3' overhangs cannot incorporate label efficiently, thus selective labeling marks fragments with appropriate co

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Digestion & Mapping

Which clone orientation produces 1.7 kb and 4.1 kb fragments on EcoRI+HindIII digestion?

Insert orientation determines relative positions of internal restriction sites to vector backbone sites. In one orientation, EcoRI and HindIII may be close together, producing one very small fragment that may be overlooked and one almost full-length band resembling empty vector. Flipping insert swaps distances, placing EcoRI site distant from HindIII site, generating 1.7 kb and 4.1 kb fragments that sum to total recombinant size of 5.8 kb. Observing these distinct sizes allows unambiguous discrimination between forward and reverse clones, critical for ensuring correct promoter-driven expressio

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Digestion & Mapping

What type of fragment is obtained from EcoRI+HindIII if both create sticky ends?

EcoRI recognizes GAATTC and HindIII recognizes AAGCTT, both cutting asymmetrically within the palindromic site to leave four-nucleotide 5' overhangs that are cohesive or sticky and complementary to themselves. When a DNA fragment is excised using these two different sticky-end cutters, each terminus retains its characteristic single-stranded extension derived from its own site. Self-ligation is prevented because ends are incompatible. Therefore the isolated insert carries two different sticky ends, one EcoRI-derived and one HindIII-derived, enabling efficient directional insertion into a simil

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details