Unequivocally the simplest #cloning technique is the Advanced QUick Assembly (#AQUAcloning) and quite literally you just need '#aqua' to do it! Although it has not been used and referred much but anything this much cost-efficient is considerable during cash-crunch, isn't it?
Principle
It has the simple principle of #assembly based cloning where the insert and vector linear DNA is incubated together in #ddH2O for 1 hour at room temperature to find the homologous regions and anneal. Subsequent transformation helps the plasmid garner benefits of bacteria's inherent in vivo enzyme processing activities.
FIGURE: A simplified protocol of the simplest cloning technique!
Protocol
PCR amplification of insert and vector: This is to be done with #overhang-tailed primers to add homologous regions to the ends of the DNA fragments.
AQUA Reaction Mix: A 10-15 μL reaction can be set up with the purified #PCR products of insert and vector in #ddH2O. The reaction is to be incubated at room temperature for 1 hour for maximum efficiency in this method.
Transformation: 5 μL of the AQUA #DNA mixture is transformed into ~50μL of commercial chemically-#competentcells.
Merits
#Seamless cloning with no scar.
No enzymes or buffers are used, virtually a #NoCost alternative.
No limits of #restrictionenzymes or #proprietaryvectors.
Demerits
Takes longer time i.e., ~1 hour to complete.
Not much efficient when #RestrictionDigested vector is used.
Applications
Multiple DNA fragment assembly.
#Insertion, #deletion mutagenesis.
#pointmutation introduction in target sequences.
Quick coupled cloning and protein expression.
More to munch on:
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