Hi Anamika,
Just to be clear, the M13 ori is for production of ssDNA using a helper phage.
Replication of that plasmid is more likely from another ColE1-derived origin
elsewhere on the vector and also likely to give high copy numbers - which it
seems it does. The p15 ori is indeed quite a low copy number origin, around
10-20 copies per cell as I remember but you can look that up. I would suggest
trying a PCR using vector- and insert-specific primers as a diagnostic in the
first instance and work from there.
Best
Steve
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Steve Smerdon
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1st Floor IBR West (WX 1.19)
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Topics of the day:
1. OFFTOPIC question "Two plasmids in one host cell"
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Date: Wed, 30 Dec 2020 11:35:24 +0200
From: Anamika Singh <anamika.ii...@gmail.com>
Subject: OFFTOPIC question "Two plasmids in one host cell"
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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