This #PIPE (Polymerase Incomplete Primer Extension) can serve piping hot and fresh #clones to you, #automatically. Yes, you can get this cloning workflow #automated and generate a #HighThroughput robust repertoire of expression clones! Dive in for a brief yet basic understanding of the same.
Principle
Conventional #PCR generates a considerably significant pool of partially single-stranded #DNA fragments resulting from incomplete #primer extension. These #DNA products have consistent 5' ends defined by the amplification primers, with staggered 3' ends of varying lengths as a function of incomplete primer extension. If primers are designed with complementary 5' ends for both insert and vector with about 14-17 bases, they may function as annealing templates for combining #PCR fragments when cloning. The annealed and combined complete plasmids are then transformed into host cells without any post-PCR enzymatic manipulations.
Protocol
Primer design: Design the primers following standard rules like, #forward primer structure is 5’-upstream vector sequence-upstream insert sequence-3’, #reverse is 5’-RC downstream vector sequence-RC downstream insert sequence-3’,
Insert/ vector PCR: In both cases follow the standard #cycle, excluding the final extension step and minimum possible template DNA as: 98C for 3mins, followed by 35 cycles of 98C for 30secs, annealing for 30secs and 72C for 45secs per kb, thereby cooling down to amenable temperature.
PIPE reaction: Combine equal volume of both the #PCR products, or in appropriate molar ratio (after quantification), along with 10U DpnI per 10uL of reaction and incubate at 37C for 1 to 3 hours.
Transformation
Clone screening
Merits
No post-PCR enzymatic manipulations.
#LowCost, doesn't require gel extractions.
Simple 2 PCR protocol, with the advantage of 2 backup strategies namely digestion-ligation and #InFusion.
Amenable to #automation.
Demerits
Might need extensive standardizations with PCR cycles.
Might become cumbersome for >10kb vectors.
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