The very foundation of #GeneticEngineering was laid with the advent of cloning, and obviously that was digestion-ligation cloning. With the discovery of a number of restriction endonucleases and their specific cut sites, it was becoming more and more easier to chop off any #DNAfragment from any source and pasting it to suitable vectors. Even today it is practiced in many labs for its #versatility.
Principle
The principle of classical cloning method revolves around restriction endonucleases that chop DNA at specific sites and ligases that form phosphodiester bond between opposing 5' phosphate and 3' hydroxyl termini. With a concerted action of these, circular plasmids harboring your gene of interest can be prepared.
Protocol
Insert/ vector Digestion: Insert and vector #DNA fragments can be digested in separate reactions with defined set of #enzymes in a compatible #buffer system. Minimum of 1ug DNA should be used for digestion keeping in mind losses at subsequent stages.
Gel extraction: Although simple, but excising the perfect band of products is really important. Run the #AgaroseGel slow for longer time, to separate the bands properly. Excise the thinnest possible #gel slice, to avoid different DNA species or clogging the column filter. (Tip: Incubate the column with wash buffer for 1 min at room temperature after passing the melted gel solution to avoid any guanidium salt carryover to your ligation step)
Quantification: Quantitate the gel extracted #DNA with #spectrophotometer. Give special attention to A260/A280 and A260/A230 ratio to set up ligation. (Tip: If A260/A230 is <2, try increasing the ligation reaction volume a bit, to mitigate inhibition of ligase).
Ligation: #Ligases catalyze the formation of #phosphodiester bond between opposing 5' phosphate and 3' hydroxyl termini. Vector and insert DNA products can be incubated with ligase in various molar ratio and temperature-time combination to achieve perfectly ligated circular #plasmids.
Transformation: Ligation mixes, after incubation can be transformed in E. coli.
Colony screening and confirmation: This can be achieved in multiple ways, colony propagation>digestion>insert dropdown, colony #PCR, colony #sequencing etc.
Merits
#Specificity of restriction enzymes impart #versatility to the system.
Apart from a large array of restriction enzymes, a number of helping enzymes, like #ShrimpAlkalinePhosphatase, #CalfIntestinalPhosphatase, #T4PolynucleotideKinase and #ligases are available.
Demerits
#unavailability of restriction sites often hamper process, although restriction sites can be inserted with PCR, it increases the cost.
As it requires a number of enzymatic manipulations, the #efficiency often takes a backseat because you cannot really make sure 100% action from all of them.
Even after using two different restriction enzymes, self-ligation of DNA fragments is not very rare.
Applications
Different recombination manipulations.
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