Hi All,
Recently I'm working on an AAV plasmid and trying to make new plasmid. And I checked on many website, some said to use stbl3 and some said use stbl2. I got confused about it and didn't see much difference between them. I know DH5a can't be used. But what's difference between stbl2 and stbl3?
Thanks. and for an update, it seems stbl3 grow much faster than stbl2, and both them working well in my unstable plasmid.lentivirus, cre-recombinase
it is recommended to use NEB stable cells, and the conclusion came from the NEB response showed as followed:
The genotype of NEB Stable is not related to the Stbl2 or Stbl3 cell line but the performance of NEB Stable is superior.
The performance of all 3 strains was tested by cloning of a DNA fragment with 5 x 32bp repeats. The test construct is named pUC-5xREP and it was created using the Gibson Assembly method of cloning. Following transformation of the Gibson Assembly reaction into the respective strain, 5xREP insert stability was analyzed by colony PCR:
Stbl2 colonies achieved 1-mm diameter in approximately 24 hrs at 30°C. This population of colonies all carried plasmid clones with deletions (33 of 33 colonies analyzed).
Stbl3 colonies achieved 1-mm diameter in 18-20 hrs at 30°C. Of 33 colonies analyzed, 22 contained full-length 5xREP insert (67% correct).
NEB Stable cells grow well at 30°C and 1-mm diameter colonies are obtained after 18-20 hrs at 30°C. 5xREP insert stability was best when using NEB Stable as the cloning strain (97% correct, 32 of 33 colonies analyzed).
Importantly, NEB Stable carries the endA mutation (in contrast to Stbl3) so that isolated plasmids are free of Endonuclease I.
Hope this will help.
I wonder what is the influence of the repeat length on the plasmid stability. I have worked with repeats of various synthetic short promoter regions (pGL4.23-based vector with a luciferase reporter) with 2-6 x 36bp, 2-16 x 86bp repeats. These plasmids grew fine in a more general cloning strain XL2-blue (also recA-minus) and I didn't notice any losses of these repeats. However, I didn't conduct a similar comparison like to do a PCR from 30 colonies to check the insert length in all of them.
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