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Plasmid Name
pFastBac His6 TEV cloning vector with BioBrick PolyPromoter LIC Subcloning (438-B)
RRID:Addgene_55219 RRID Copied  
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RRID:Addgene_55219
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Plasmid Information

URL: http://www.addgene.org/55219

Proper Citation: RRID:Addgene_55219

Bacterial Resistance: Ampicillin

Defining Citation: PMID:28668116

Vector Backbone Description: Vector Backbone:pFastBac; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin

Comments: In order to increase the efficiency of subcloning with the Series-11 Macrobac plasmids we developed a new strategy the uses LIC (Ligation Independent Cloning). The problem with the Series-11 ligation mediated subcloning was that when the plasmid size approached or exceeded 20 kb we had to screen many colonies to find a positive. We developed a strategy to use LIC for subcloning. Because this method uses no ligase, the cloning background associated with re-ligation of the empty plasmid are eliminated. 438-B has a TEV cleavable His6 at the N-terminus. The 438-B vector use the LICv1 Forward and Reverse primers. LICv1Forward - 5'-TACTTCCAATCCAATGCA-3' LICv1 Reverse - 5'-TTATCCACTTCCAATGTTATTA-3' As the target plasmid size gets >20kb you may have to increase the amount of DNA you anneal and/or transform. We have found this method gives no background colonies at all and 100% of the colonies we pick are positive. The cells we transform into are XL1Blues with a competency ~6x10^7cfu/ul. For more information, please see our website: http://qb3.berkeley.edu/qb3/macrolab/

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Data and Source Information

Source: Addgene