Purpose(Empty Backbone) GST-tev-yORF
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||97092||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size (bp) 6130
Vector typeInsect Expression
- Promoter Polyhedrin promoter
/ Fusion Proteins
- GST tag (N terminal on backbone)
- TEV cleavage site (N terminal on backbone)
Growth in Bacteria
Copy numberHigh Copy
- Cloning method Ligation Independent Cloning
- 5′ sequencing primer GST 3' F (5' ACTTCATGTTGTATGACGCTC 3')
- 3′ sequencing primer LicBac dual V1 Rv (5'-caggttcagggggaggtgtg-3') (Common Sequencing Primers)
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-GST has a TEV cleavable GST at the N-terminus. MBP can improve expression and solubility of the target protein. The 438-GST 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/
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:438-GST was a gift from Chris Jeans (Addgene plasmid # 97092 ; http://n2t.net/addgene:97092 ; RRID:Addgene_97092)