PurposeBacterial expression of Pfu DNA polymerase
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||12509||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 5711
Vector typeBacterial Expression
Growth in Bacteria
Copy numberHigh Copy
Gene/Insert namePfu DNA polymerase
Insert Size (bp)2325
MutationY218H, Q462R, F533L, K559I and V683A (mutations were not intentional, but plasmid functions as described)
- Cloning method Restriction Enzyme
- 5′ cloning site NdeI (not destroyed)
- 3′ cloning site XhoI (not destroyed)
- 5′ sequencing primer TAATACGACTCACTATAGGG
- 3′ sequencing primer GCTAGTTATTGCTCAGCGG (Common Sequencing Primers)
Please note that the plasmid pET16B.Pfu contains an Ampicillin resistance marker, and the BL21 bacterial strain contains a plasmid, pSJS1240, that confers spectinomycin resistance and allows for expression of genes with rare codons.
Addgene provides this plasmid in the DH5alpha bacterial strain. For expression, please transform BL21(DE3)Star bacterial cells with this plasmid and include pSJS1240 (Addgene plasmid number 12234), if necessary for expression of rare codons.
Addgene's sequencing results found Y218H, Q462R, F533L, K559I and V683A mutations in the Pfu sequence when compared to GenBank entry D12983. These mutations were not intentional, but plasmid functions as described in the associated publication listed below.
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:pET16B.Pfu was a gift from Sung-Hou Kim (Addgene plasmid # 12509 ; http://n2t.net/addgene:12509 ; RRID:Addgene_12509)
For your References section:Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase. Goldman S, Kim R, Hung LW, Jancarik J, Kim SH. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1. 54(Pt 5):986-8. 10.1107/S0907444998000353 PubMed 9757114