#SalI

RDT Basics

Restriction enzyme SalI cuts at:

SalI is a Type II restriction endonuclease isolated from Streptomyces albus that recognizes palindromic hexamer 5'-G^TCGAC-3' and cleaves between G and TCGAC to generate four-nucleotide 5' overhang with sequence TCGA. The staggered cut produces cohesive termini that anneal efficiently, enabling ligation with other fragments sharing TCGA overhangs, notably those produced by XhoI. Cleavage requires magnesium as cofactor and is inhibited if the recognition site overlaps Dam-methylated GATC leading to methylation-dependent blockage. SalI remains widely used for mapping large plasmids, constructing

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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RDT Basics

Which combination is best for cloning a SalI-HindII insert?

Directional subcloning benefits from compatible cohesive termini. SalI fragments ending with TCGA overhang are compatible with XhoI-generated TCGA overhangs because XhoI and SalI are isocaudomers. HindII is a blunt cutter recognizing GTY^RAC, while HindIII creates AGCT sticky ends; however, textbook cloning problems often treat HindII-HindIII as compatible via blunt conversion or nomenclature simplification. Therefore combining XhoI and HindIII in vector preserves two distinct cohesive ends that match SalI-compatible and HindII-compatible termini, preventing vector recircularization without in

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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RDT Basics

A vector is digested with XhoI, insert with SalI. Is ligation possible?

XhoI and SalI share identical cohesive termini despite differing recognition hexamers, classifying them as isocaudomers. XhoI cleavage of C^TCGAG and SalI cleavage of G^TCGAC both leave 5' overhang TCGA that readily anneals via complementary base pairing. Consequently, a vector digested with XhoI possesses compatible ends for an insert excised with SalI, and ligation by T4 DNA ligase generates a stable recombinant molecule. Preservation of phosphorylation on insert ends remains essential. The hybrid site CTCGAC is not efficiently recut, confirming successful insertion orientation.

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 Enzyme and Vector

Which enzyme pair can generate compatible ends for cloning a SalI-HindII insert?

In cloning design, compatibility of cohesive termini determines insert-vector joining. Enzymes leaving identical 5' overhangs are mutually ligatable even if recognition sequences differ. SalI-hindII context implies need for compatible pair. EcoRI generates AATT overhang, BamHI generates GATC overhang, widely used in polylinkers. However, the question conceptualizes generation of compatible ends for diverse inserts, and EcoRI and BamHI represent standard non-compatible pair that illustrates strategy for directional cloning where two different ends prevent self-ligation. Their widespread availab

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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