New England Biolabs Inc
NEBuilder HiFi DNA Assembly Master Mix
E2621G
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Two PCR fragments flanking the region of desired gene to be deleted in E. coli were assembled into plasmid vector of 4 kb size using NEB HiFi DNA assembly master mix and assembled product was transformed into in-house competent cells by electroporation. The efficiency of NEB HiFi assembly was much better compared to other methods and got desired clones after transformation.
Creation of gene knock outs in E.coli
Bacterial DNA and plasmid DNA
550 bp PCR fragments flanking up and down stream of the desired gene to be deleted were amplified using Q5 high fidelity polymerase. Also, plasmid DNA of size 4 kb was amplified in a PCR using Q5 hihg fidelity DNA polymerase. 2 inserts and vector fragments were assembled using NEBuilder HiFi assembly master mix in 20 microliter reaction with vector:insert ratio of 1:2 and 0.02 pmoles of DNA for 3 fragment assembly. Assembled mix was transformed into recipient competent E.coli cells. Desired clones ware selected on the basis of selectable marker and confirmed by sequencing.
Design overlapping PCR primers for amplifying PCR fragments and as well for vector to get successful assembly.
Assembly for 3 fragments was successful and got desired deletion of gene in E.coli strain during my experiments.
None
Effective and no hassle of restriction digestion and additional steps
Slightly expensive compared to traditional cloning methods
Saves a lot of time and especially good for difficult clone (deletion/insertion) genes.
Product Name: NEBuilder® HiFi DNA Assembly Master MixSupplier Page