The pACYC vectors (probably your p15ori plasmid) is said to have a copy
number of ~10 copies per cell while your other plasmid is likely in the
hundreds - see:
https://blog.addgene.org/plasmid-101-origin-of-replication
The protein expression level from the lower copy number plasmid is
usually a bit lower, but not always.
For sequencing, we use primers that are specific to the pACYC vector so
it does not matter if there is a second higher copy plasmid present in
the miniprep.
To increase the amount of pACYC vector for sequencing, you can always
amplify the copy number by adding spectinomycin to a growing culture -
this inhibits protein synthesis, but the low copy plasmids continue to
replicate to hundreds of copies per cell.
Darren
On 30/12/2020 10:35, Anamika Singh wrote:
Hi All,
I have two constructs having different ori, p15ori and M13 ori,
different promoters araBAD promoter and LacI, and different antibiotic
resistance chloramphenicol and Ampicillin respectively.
I am managed to get the transformants and getting the expected result
after blunt digestion with the EcoRV enzyme. Since both the plasmids
have the site for EcoRV. But the p15 ori has a low copy number that's
why I am seeing the very faint band as compared to other plasmid in
the sample.
So I would like to know is there any way that I can quantify the low
copy number plasmid.
Because I am not able to sequence it with the specific primers it
could be due to its low concentration.
Please advise.
Thank you.
--
Dr. Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036
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**********************************************************************
Dr. Darren J. Hart,
CNRS Research Director, Institut de Biologie Structurale (IBS)
Unité Mixte de Recherche UMR5075 (CEA-CNRS-UGA)
Director, Integrated Structural Biology Grenoble (ISBG)
Unité Mixte de Service UMS3518 (CNRS-UGA-CEA-EMBL)
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Email: darren.h...@ibs.fr
Tel: +33 4 57 42 85 86
Physical address: IBS/ISBG, 71 avenue des Martyrs, 38000 Grenoble, France
Postal address: IBS/ISBG, 71 avenue des Martyrs, CS 20192, 38042
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