I am planning to overexpress an sRNA in K.pneumoniae (inherently resistant to Amp, hence cannot use pUC19 because of it Amp resistance and not useful to scree). Which is better to clone in an expression vector? A gblock which can be directly pasted in or an ultramer? Sorry I am being too ignorant/silly. This is my first time cloning an sRNA and I am totally confused. Thank you very much in advance for any suggestions on this.
Ultramers are apparently ≤200 bp and can either be single or double stranded. gBlocks are ≥125 bp and are always double stranded. There's also a price difference, gBlocks aren't priced by exact base pair number but rather by what range they fall into, which makes them significantly cheaper than ultramers at larger sizes but potentially more expensive if you're close to the minimum required size.
Which one you would use ultimately depends on the size of the fragment you need, keeping in mind if you need an additional sequence for a promoter/terminator/homologous ends for Gibson assembly, etc.
As a comment to your statement about being unable to use Amp due to inherent resistance, my lab worked on Serratia for years which also showed similar resistance. If you increase the Amp concentration then the selection works. We used 500ug/ml of ampicillin and the selections were clean and we could select for pUC plasmids with transformation.
Alexandra Johnson Thank you very much for the suggestion, I have now added a T7 promoter and an operator (lac) infront of an sRNA sequence followed by T7 terminator and ordered for a gblock. Hope it works.
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