I recently purified 18mL total volume after dialysis of Taq polymerase that is active up to half dilution. However, my PI swears we could produce more than that (we used to have an old plasmid with a protocol that anecdotally produced up to 40 dilutions of active Taq). I am trying to optimize this protocol to yield more sample.
Our culture conditions include induction at OD=0.4 and add 1mM IPTG and for 16 hrs at 37C.
The original protocol from Addgene indicates induction at OD=0.2. My experience with other proteins is induction at OD=0.6-0.8 for 3 hrs at 37C or 16-20hrs at 20C.
Your suggestions will be greatly appreciated!
- PURPOSEBacterial expression of Taq polymerase
- DEPOSITING LABDavid Engelke
- PUBLICATIONEngelke et al Anal Biochem. 1990 Dec . 191(2):396-400. ( How to cite )
- SEQUENCE INFORMATION Sequences (3)
ORDERING ItemCatalog #DescriptionQuantityPrice (USD)Plasmid25712Standard format: Plasmid sent in bacteria as agar stab1$75Add to CartThis material is available to academics and nonprofits only.
BACKBONE Vector backbonepTTQ18 (Search Vector Database)Backbone size w/o insert (bp)4563 Bacterial Expression Vector type
GROWTH IN BACTERIA Ampicillin Bacterial Resistance(s)37°C Growth TemperatureDH5alpha Growth Strain(s)High Copy Copy number
GENE/INSERT Taq polymerase Gene/Insert nameThermus aquaticus Species2501 Insert Size (bp)
(Common Sequencing Primers)CLONING INFORMATION Cloning methodRestriction Enzyme 5′ cloning siteEcoRI (not destroyed) 3′ cloning siteBglII (unknown if destroyed) 5′ sequencing primerTac promoter 3′ sequencing primerM13_puc_F
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