#HotFusionCloning has marked its space in the #cloning arena with remarkable efficiency even for cloning in fragments with inverted repeats aimed at applications such as #RNAi. It is basically a modified version of #GibsonAssembly. Read to find out its approach.
Principle
This is a one-pot one-step reaction at 50°C for 1 hour. The reaction buffer contains: #T5exonuclease and #PhusionDNApolymerase. #T5exonuclease removes nucleotides from the 5' to 3' end leaving single strand tails of 3' overhangs that are complementary and anneal to single strand tails of the adjacent fragment or vector generated by the #T5exonuclease in the same reaction. #PhusionDNApolymerase fills in gaps that are over-generated by #T5exonuclease. Annealed fragments are transformed and the nick sites in the nucleotide chain are repaired in vivo by E. coli.
Protocol
5X Pre-assembly Buffer (0.5 M #Tris pH 7.5, 50 mM #MgCl2, 1 mM each #dNTP, 50 mM #DTT, 25% #PEG8000)
2X Hot Fusion Buffer (For 400uL, 160uL 5X Pre-assembly Buffer, 0.3uL of 10U/uL T5 exonuclease [0.0075 U/uL], 10uL of 2U/uL #Phusion polymerase [0.05 U/uL], 230uL dH2O)
Hot Fusion Reaction: 30-50ng linearized vector, 90-300 ng insert #PCR product, 10uL of 2X Hot Fusion Buffer, dH2O upto 20uL reaction volume. reaction mix is incubated for 1 hour at 50°C, then temperature is slowly ramped down to 20°C in 5 minutes (0.1°C per second), and held at 10°C.
Transformation: Transform 2.5uL of the reaction into 50uL of highly competent E. coli cells of your choice. Follow your standard #transformation protocol, plate the cells with appropriate antibiotic and incubate at 37C overnight.
Colony PCR/ Sequencing: Among the colonies >95% are expected to be transformants. This can be validated through colony PCR with one vector-specific and one insert-specific primer. Colony sequencing may also be done with those sets of primers.
Merits
This method is #directional and independent of any #restriction sites.
Highly #efficient (efficiency is in the order of 90–95%) even in gene fragments with inverted repeats.
It is a #HighThroughput method.
Demerits
This might not be as efficient in cases of multi-fragment assembly.
Applications
More to much on:
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